DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF CRISPR SYSTEMS AND COMPOSITIONS FOR GENOME EDITING AS TO HEMATOPOIETIC STEM CELLS (HSCs)

ABSTRACT

The invention provides for delivery, engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are delivery systems and tissues or organ which are targeted as sites for delivery. Also provided are vectors and vector systems some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity and to edit or modify a target site in a genomic locus of interest to alter or improve the status of a disease or a condition.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation-in-part of international patentapplication Serial No. PCT/US2015/065405 filed Dec. 11, 2015 andpublished as PCT Publication No. WO 2016/094880 on Jun. 16, 2016 andwhich claims priority from U.S. application Ser. No. 62/091,461 filedDec. 12, 2014. Reference is made to PCT/US2014/070127 filed Dec. 12,2014, which claims the benefit of U.S. provisional patent applications61/915,176; 61/915,192; 61/915,215; 61/915,107; 61/915,145; 61/915,148;and 61/915,153 each filed Dec. 12, 2013. Reference is made to U.S.patent application Ser. No. 14/705,719 filed May 6, 2015, which is acontinuation of PCT/US2014/070057 filed Dec. 12, 2014 and claims thebenefit of U.S. provisional patent applications 61/915,118, 61/915,148and 61/915,215 each filed Dec. 12, 2013, U.S. provisional patentapplication 62/010,441 filed Jun. 10, 2014, and U.S. provisional patentapplication 62/054,490 filed Sep. 24, 2014.

Reference is also made to US provisional patent application Serial Nos.62/181,453 filed Jun. 18, 2015, 62/207,312 filed Aug. 19, 2015,62/237,360 filed Oct. 5, 2015, and 62/255,256 filed Nov. 13, 2015,entitled CRISPR ENZYME MUTATIONS REDUCING OFF-TARGET EFFECTS.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appin cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos.MH100706, MH110049 and DK097768 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 22, 2016, isnamed 47627_99_2005 SL.txt and is 12,616 bytes in size.

FIELD OF THE INVENTION

The present invention generally relates to the delivery, engineering,optimization and therapeutic applications of systems, methods, andcompositions used for the control of gene expression involving sequencetargeting, such as genome perturbation or gene-editing, that relate toClustered Regularly Interspaced Short Palindromic Repeats (CRISPR) andcomponents thereof. The present invention relates to in vitro, ex vivoand/or in vivo systems, methods, and compositions for delivery of theCRISPR-Cas system(s) to achieve therapeutic benefits via genome editingin animals, including mammals.

BACKGROUND OF THE INVENTION

Recent advances in genome sequencing techniques and analysis methodshave significantly accelerated the ability to catalog and map geneticfactors associated with a diverse range of biological functions anddiseases. Precise genome targeting technologies are needed to enablesystematic reverse engineering of causal genetic variations by allowingselective perturbation of individual genetic elements, as well as toadvance synthetic biology, biotechnological, and medical applications.Although genome-editing techniques such as designer zinc fingers (ZFN),transcription activator-like effectors (TALEs), or homing meganucleasesare available for producing targeted genome perturbations, there remainsa need for new genome engineering technologies that are affordable, easyto set up, scalable, and amenable to targeting multiple positions withinthe eukaryotic genome.

SUMMARY OF THE INVENTION

Despite valid therapeutic hypotheses and strong efforts in drugdevelopment, there have only been a limited number of successes usingsmall molecules to treat diseases with strong genetic contributions.Thus, there exists a pressing need for alternative and robust systemsfor therapeutic strategies that are able to modify nucleic acids withindisease-affected cells and tissues. Adding CRISPR-Cas system(s) to therepertoire of therapeutic genome engineering methods significantlysimplifies the methodology and accelerates the ability to catalog andmap genetic factors associated with a diverse range of biologicalfunctions and diseases, develop animal models for genetic diseases, anddevelop safe, effective therapeutic alternatives. To utilize theCRISPR-Cas system(s) effectively for genome editing without deleteriouseffects, it is critical to understand aspects of engineering,optimization and cell-type/tissue/organ specific delivery of thesegenome engineering tools, which are aspects of the claimed invention.Aspects of this invention address this need and provide relatedadvantages.

An exemplary CRISPR complex may comprise a CRISPR enzyme protein (e.g.,Cas9) complexed with a guide sequence hybridized to a target sequencewithin the target polynucleotide. The guide sequence is linked to atracr mate sequence, which in turn hybridizes to a tracr sequence.Applicants have optimized components of the CRISPR-Cas genomeengineering system, including using SaCas9 (Cas9 from Staphylococcusaureus). SaCas9 is preferred. Reference is made to PCT applicationPCT/US2014/070152, filed Dec. 12, 2014, entitled, “ENGINEERING OFSYSTEMS, METHODS AND OPTIMIZED GUIDE COMPOSITIONS WITH NEW ARCHITECTURESFOR SEQUENCE MANIPULATION” (the “New Architecture PCT”), published asWO/2015/089473, incorporated herein by reference, which involves SaCas9.For instance, the New Architecture PCT involves provides a compositionwhich is a non-naturally occurring or engineered CRISPR-Cas system dualguide RNA molecule (dgRNA) capable of effecting the manipulation of atarget nucleic acid within a prokaryotic or eukaryotic cell when incomplex within the cell with a CRISPR enzyme comprising a Staphyloccocusaureus Cas9 enzyme (SaCas9); the dgRNA comprising: I. a chimeric RNAmolecule comprising, in a tandem arrangement: a) a guide sequence, whichis capable of hybridizing to a sequence of a target nucleic acid to bemanipulated; and b) a tracr mate sequence, comprising a region of sensesequence; and II. a tracr RNA molecule, comprising a region of antisensesequence which is capable of hybridizing with the region of sensesequence of the tracr mate sequence; wherein the guide sequencecomprises a length of 21 or more nucleotides. The New Architecture PCTalso involves a non-naturally occurring or engineered CRISPR-Cas systemchimeric single guide RNA molecule (sgRNA) capable of effecting themanipulation of a target nucleic acid within a prokaryotic or eukaryoticcell when in complex within the cell with a CRISPR enzyme comprising aCRISPR enzyme, or a non-naturally occurring or engineered compositioncomprising a CRISPR-Cas system comprising said sgRNA, the sgRNAcomprising a guide sequence capable of hybridizing to a target sequencein a locus, e.g., genomic locus of interest in a cell whereinarchitecture of the sgRNA is modified. The New Architecture PCT alsoinvolves a non-naturally occurring or engineered CRISPR-Cas systemchimeric single guide RNA molecule (sgRNA) capable of effecting themanipulation of a target nucleic acid within a prokaryotic or eukaryoticcell when in complex within the cell with a CRISPR enzyme comprising aStaphyloccocus aureus Cas9 enzyme (SaCas9); the sgRNA comprising, in atandem arrangement: I. a guide sequence, which is capable of hybridizingto a sequence of the target nucleic acid to be manipulated; II. a tracrmate sequence, comprising a region of sense sequence; III. a linkersequence; and IV. a tracr sequence, comprising a region of antisensesequence which is positioned adjacent the linker sequence and which iscapable of hybridizing with the region of sense sequence thereby forminga stem-loop; wherein the guide sequence comprises a length of 21 or morenucleotides. The dual guide RNA molecules, the dgRNAs, sgRNAs, DNApolynucleotide molecules, DNA expression vectors, delivery vectors,methods, systems, compositions or complexes of the New Architecture PCT,especially such comprising a Staphyloccocus aureus Cas9 enzyme (SaCas9)or a fragment or derivative of Staphyloccocus aureus Cas9 (e.g., such asan SaCas9 having an amino acid substitution or mutation such as N580A)are envisioned as being useful in the practice of the present invention;and, it is noted that in an aspect in the dgRNA or sgRNA the guidesequence can comprise a length of 22 or more nucleotides, 23 or morenucleotides, or 24 or more nucleotides, preferably wherein the guidesequence comprises a length of 21, 22, 23 or 24 nucleotides; and thelinker sequence can commences after position +36 or less of the sensesequence, after position +25 or less of the sense sequence, or afterposition +21 or less of the sense sequence, or after position +18 orless of the sense sequence, or after position +15 or less of the sensesequence, or after position +14 or less of the sense sequence; or in thedgRNA the the tracr mate has a length of 36 nucleotides or less, or hasa length of 25 nucleotides or less, or has a length of 21 nucleotides orless, or has a length of 18 nucleotides or less, or has a length of 15nucleotides or less, or has a length of 14 nucleotides or less.

Various delivery means may be employed for delivering components of theCRISPR-Cas system to cells, tissues and organs, ex vivo and/or in vivo.Applicants have effectively packaged CRISPR-Cas system components (e.g.,comprising SaCas9) into a viral delivery vector, e.g., AAV, and havedemonstrated that it can be used to modify endogenous genome sequence inmammalian cells in vivo. Moreover, Applicants have modified HSCs with aparticle system. A key feature of Applicants' present invention it thatit effectively addresses the challenges of low efficiency of in vivodelivery (of therapeutic components) and low efficiency of homologydirected repair (HDR) and in particular challenges associated withco-delivery are solved by the small Cas9, SaCas9 from Staphylococcusaureus, which can be readily packaged into a single Adeno-associatedvirus (AAV) vector to express both the Cas9 protein and itscorresponding sgRNA(s). Further, importantly, Applicants have shown thatintroduction of small SaCas9, has reduced the number of viral vectorsrequired to perform HDR from 3 vectors to 2 vectors. And the number ofparticles to be contacted with HSCs can be one or two. In one aspect,the invention provides methods for using one or more elements of aCRISPR-Cas system. The CRISPR complex of the invention provides aneffective means for modifying a target polynucleotide in a locus orloci, e.g., genomic locus or loci of an HSC. The CRISPR complex of theinvention has a wide variety of utilities including modifying (e.g.,deleting, inserting, translocating, inactivating, activating) a targetpolynucleotide in an HSC. As such the CRISPR complex of the inventionhas a broad spectrum of applications in, e.g., gene or genome editing,gene therapy, drug discovery, drug screening, disease diagnosis, andprognosis. Aspects of the invention relate to Cas9 enzymes havingimproved targeting specificity in a CRISPR-Cas9 system having guide RNAshaving optimal activity, smaller in length than wild-type Cas9 enzymesand nucleic acid molecules coding therefor, and chimeric Cas9 enzymes,as well as methods of improving the target specificity of a Cas9 enzymeor of designing a CRISPR-Cas9 system comprising designing or preparingguide RNAs having optimal activity and/or selecting or preparing a Cas9enzyme having a smaller size or length than wild-type Cas9 wherebypackaging a nucleic acid coding therefor into a delivery vector is moreadvanced as there is less coding therefor in the delivery vector thanfor wild-type Cas9, and/or generating chimeric Cas9 enzymes. Alsoprovided are uses of the present sequences, vectors, enzymes or systems,in medicine. Also provided are uses of the same in gene or genomeediting.

In the invention, the Cas enzyme can be wildtype Cas9 including anynaturally-occurring bacterial Cas9. Cas9 orthologs typically share thegeneral organization of 3-4 RuvC domains and a HNH domain. The 5′ mostRuvC domain cleaves the non-complementary strand, and the HNH domaincleaves the complementary strand. All notations are in reference to theguide sequence. The catalytic residue in the 5′ RuvC domain isidentified through homology comparison of the Cas9 of interest withother Cas9 orthologs (from S. pyogenes type II CRISPR locus, S.thermophilus CRISPR locus 1, S. thermophilus CRISPR locus 3, andFranciscilla novicida type II CRISPR locus), and the conserved Aspresidue (D10) is mutated to alanine to convert Cas9 into acomplementary-strand nicking enzyme. Similarly, the conserved His andAsn residues in the HNH domains are mutated to Alanine to convert Cas9into a non-complementary-strand nicking enzyme. In some embodiments,both sets of mutations may be made, to convert Cas9 into a non-cuttingenzyme. Accordingly, the Cas enzyme can be wildtype Cas9 including anynaturally-occurring bacterial Cas9. The CRISPR, Cas or Cas9 enzyme canbe codon optimized for HSC, or a modified version, including anychimaeras, mutants, homologs or orthologs. In an additional aspect ofthe invention, a Cas9 enzyme may comprise one or more mutations and maybe used as a generic DNA binding protein with or without fusion to afunctional domain. The mutations may be artificially introducedmutations or gain- or loss-of-function mutations. The mutations mayinclude but are not limited to mutations in one of the catalytic domains(D10 and H840) in the RuvC and HNH catalytic domains, respectively.Further mutations have been characterized. In one aspect of theinvention, the transcriptional activation domain may be VP64. In otheraspects of the invention, the transcriptional repressor domain may beKRAB or SID4X. Other aspects of the invention relate to the mutated Cas9 enzyme being fused to domains which include but are not limited to anuclease, a transcriptional activator, repressor, a recombinase, atransposase, a histone remodeler, a demethylase, a DNAmethyltransferase, a cryptochrome, a light inducible/controllable domainor a chemically inducible/controllable domain. The invention can involvesgRNAs or tracrRNAs or guide or chimeric guide sequences that allow forenhancing performance of these RNAs in cells. The CRISPR enzyme can be atype I or III CRISPR enzyme, preferably a type II CRISPR enzyme. Thistype II CRISPR enzyme may be any Cas enzyme. A preferred Cas enzyme maybe identified as Cas9 as this can refer to the general class of enzymesthat share homology to the biggest nuclease with multiple nucleasedomains from the type II CRISPR system. Most preferably, the Cas9 enzymeis from, or is derived from, spCas9 or saCas9. By derived, Applicantsmean that the derived enzyme is largely based, in the sense of having ahigh degree of sequence homology with, a wildtype enzyme, but that ithas been mutated (modified) in some way as described herein

It will be appreciated that the terms Cas and CRISPR enzyme aregenerally used herein interchangeably, unless otherwise apparent. Asmentioned above, many of the residue numberings used herein refer to theCas9 enzyme from the type II CRISPR locus in Streptococcus pyogenes.However, it will be appreciated that this invention includes many moreCas9s from other species of microbes, such as SpCas9, SaCas9, St1Cas9and so forth. Further examples are provided herein. The skilled personwill be able to determine appropriate corresponding residues in Cas9enzymes other than SpCas9 by comparison of the relevant amino acidsequences. Thus, where a specific amino acid replacement is referred tousing the SpCas9 numbering, then, unless the context makes it apparentthis is not intended to refer to other Cas9 enzymes, the disclosure isintended to encompass corresponding modifications in other Cas9 enzymes.An example of a codon optimized sequence, in this instance optimized forhumans (i.e. being optimized for expression in humans) is providedherein, see the SaCas9 human codon optimized sequence. Whilst this ispreferred, it will be appreciated that other examples are possible andcodon optimization for a host species is known. The inventioncomprehends methods wherein the Cas9 is a chimeric Cas9 proteins. Thesemethods may comprise N-terminal fragment(s) of one Cas9 homolog withC-terminal fragment(s) of one or more other or another Cas9 homolog. Itwill be appreciated that in the present methods, where the organism isan animal, the modification may occur ex vivo or in vitro, for instancein a cell culture and in some instances not in vivo. In otherembodiments, it may occur in vivo. The invention comprehends in someembodiments a composition of the invention or a CRISPR enzyme thereof(including or alternatively mRNA encoding the CRISPR enzyme), whereinthe target sequence is flanked at its 3′ end by a PAM (protospaceradjacent motif) sequence comprising 5′-motif, especially where the Cas9is (or is derived from) S. pyogenes or S. aureus Cas9. For example, asuitable PAM is 5′-NRG or 5′-NNGRR (where N is any Nucleotide) forSpCas9 or SaCas9 enzymes (or derived enzymes). It will be appreciatedthat SpCas9 or SaCas9 are those from or derived from S. pyogenes or S.aureus Cas9.

The skilled person, from this disclosure and the knowledge in the art,can employ such proteins in the practice of the invention. For instance,the skilled person will be able to determine appropriate correspondingresidues in CRISPR-Cas9 enzymes from the disclosure herein and theknowledge in the art for comparison of relevant amino acid sequences.For example, the skilled person will be able to determine appropriatecorresponding residues in Cas9 enzymes other than SpCas9 by comparisonof the relevant amino acid sequences. Thus, where a specific amino acidreplacement is referred to using the SpCas9 numbering, then, unless thecontext makes it apparent this is not intended to refer to other Cas9enzymes, the disclosure is intended to encompass correspondingmodifications in other Cas9 enzymes. An example of a codon optimizedsequence, in this instance optimized for humans (i.e. being optimizedfor expression in humans) is provided herein, see the SaCas9 human codonoptimized sequence. Whilst this is preferred, it will be appreciatedthat other examples are possible and codon optimization for a hostspecies is known. The invention comprehends methods wherein theCRISPR-Cas protein or Cas9 is a chimeric, e.g., a chimeric of two ormore different CRISPR-Cas proteins or different Cas9 proteins. Thesemethods may comprise N-terminal fragment(s) of one CRISPR-Cas protein orCas9 homolog with C-terminal fragment(s) of one or more other or anotherCRISPR-Cas proteins or Cas9 homolog. It will be appreciated that in thepresent methods, where the organism is an animal, the modification mayoccur ex vivo or in vitro, for instance in a cell culture and in someinstances not in vivo. In other embodiments, it may occur in vivo. Theinvention comprehends in some embodiments a composition of the inventionor a CRISPR enzyme or protein thereof (including or alternatively mRNAencoding the CRISPR enzyme or protein), wherein the target sequence isflanked at its 3′ end by a PAM (protospacer adjacent motif) sequencecomprising 5′-motif, especially where the Cas9 is (or is derived from)S. pyogenes or S. aureus Cas9. For example, a suitable PAM is 5′-NRG or5′-NNGRR (where N is any Nucleotide) for SpCas9 or SaCas9 enzymes (orderived enzymes). It will be appreciated that SpCas9 or SaCas9 are thosefrom or derived from S. pyogenes or S. aureus Cas9.

In one aspect, the invention provides a method of modifying an organismor a non-human organism by manipulation of a target sequence in a locus,e.g., genomic locus of interest of a HSC, e.g., wherein the locus orgenomic locus of interest is associated with a mutation associated withan aberrant protein expression or with a disease condition or state,comprising:

-   -   delivering to an HSC via a delivery system, e.g., via contacting        an HSC with a particle containing, and/or one or more vectors        expressing and/or having transcription and/or translation of,        and/or one or more nucleic acid molecules (RNA, e.g., mRNA,        and/or DNA giving rise to expression and/or transcription and/or        translation in vivo in the HSC) a non-naturally occurring or        engineered composition comprising:        -   I. a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide            sequence, comprising:            -   (a) a guide sequence capable of hybridizing to a target                sequence in a HSC,            -   (b) a tracr mate sequence, and            -   (c) a tracr sequence, and        -   II. a CRISPR enzyme, optionally comprising at least one or            more nuclear localization sequences,

wherein the tracr mate sequence hybridizes to the tracr sequence and theguide sequence directs sequence-specific binding of a CRISPR complex tothe target sequence, and

wherein the CRISPR complex comprises the CRISPR enzyme complexed with(1) the guide sequence that is hybridized to the target sequence, and(2) the tracr mate sequence that is hybridized to the tracr sequence;

and

the method may optionally include also delivering a HDR template, e.g.,via the particle contacting the HSC containing or contacting the HSCwith another particle containing, the HDR template wherein the HDRtemplate provides expression of a normal or less aberrant form of theprotein; wherein “normal” is as to wild type, and “aberrant” can be aprotein expression that gives rise to a condition or disease state; and

optionally the method may include isolating or obtaining HSC from theorganism or non-human organism, optionally expanding the HSC population,performing contacting of the particle(s) with the HSC to obtain amodified HSC population, optionally expanding the population of modifiedHSCs, and optionally administering modified HSCs to the organism ornon-human organism.

The teachings provided herein provide effective methods for providingmodified hematopoietic stem cells and progeny thereof, including but notlimited to cells of the myeloid and lymphoid lineages of blood,including T cells, B cells, monocytes, macrophages, neutrophils,basophils, eosinophils, erythrocytes, dendritic cells, andmegakaryocytes or platelets, and natural killer cells and theirprecursors and progenitors. Such cells can be modified by knocking out,knocking in, or otherwise modulating targets, for example to remove ormodulate CD52 as described above, and other targets, such as, withoutlimitation, CXCR4, and PD-1. Thus compositions, cells, and method of theinvention can be used to can be to modulate immune responses and totreat, without limitation, malignancies, viral infections, and immunedisorders.

In one aspect, the invention provides a method of modifying an organismor a non-human organism by manipulation of a target sequence in locus ofinterest, e.g., a genomic locus of interest of a HSC, e.g., wherein thelocus of interest, e.g., genomic locus of interest is associated with amutation associated with an aberrant protein expression or with adisease condition or state, comprising: delivering to an HSC, e.g., viaa delivery system, e.g., via contacting an HSC with a particlecontaining, and/or one or more vectors expressing and/or havingtranscription and/or translation of, and/or one or more nucleic acidmolecules (RNA, e.g., mRNA, and/or DNA giving rise to expression and/ortranscription and/or translation in vivo in the HSC), a non-naturallyoccurring or engineered composition comprising: I. (a) a guide sequencecapable of hybridizing to a target sequence in a HSC, and (b) at leastone or more tracr mate sequences, II. a CRISPR enzyme optionally havingone or more NLSs, and III. a polynucleotide sequence comprising a tracrsequence, wherein the tracr mate sequence hybridizes to the tracrsequence and the guide sequence directs sequence-specific binding of aCRISPR complex to the target sequence, and wherein the CRISPR complexcomprises the CRISPR enzyme complexed with (1) the guide sequence thatis hybridized to the target sequence, and (2) the tracr mate sequencethat is hybridized to the tracr sequence; and

the method may optionally include also delivering a HDR template, e.g.,via the particle contacting the HSC containing or contacting the HSCwith another particle containing, the HDR template wherein the HDRtemplate provides expression of a normal or less aberrant form of theprotein; wherein “normal” is as to wild type, and “aberrant” can be aprotein expression that gives rise to a condition or disease state; and

optionally the method may include isolating or obtaining HSC from theorganism or non-human organism, optionally expanding the HSC population,performing contacting of the particle(s) with the HSC to obtain amodified HSC population, optionally expanding the population of modifiedHSCs, and optionally administering modified HSCs to the organism ornon-human organism.

The delivery system can be of one or more polynucleotides encoding anyone or more or all of the CRISPR-complex, advantageously linked to oneor more regulatory elements for in vivo expression, e.g. viaparticle(s), containing a vector containing the polynucleotide(s)operably linked to the regulatory element(s). Any or all of thepolynucleotide sequence encoding a CRISPR enzyme or protein, and RNAtherefor, e.g., guide sequence, and optionally where applicable tracrmate sequence or tracr sequence, may be RNA. It will be appreciated thatwhere reference is made to a polynucleotide, which is RNA and is said to‘comprise’ a feature such as for example, a tracr mate sequence, the RNAsequence includes the feature. Where the polynucleotide is DNA and issaid to comprise a feature such as for example, a tracr mate sequence,the DNA sequence is or can be transcribed into the RNA including thefeature at issue. Where the feature is a protein, such as the CRISPRenzyme, the DNA or RNA sequence referred to is, or can be, translated(and in the case of DNA transcribed first).

In certain embodiments the invention provides a method of modifying anorganism, e.g., mammal including human or a non-human mammal or organismby manipulation of a target sequence in an HSC e.g., a genomic locus ofinterest, such as wherein the target sequence or genomic locus ofinterest is associated with a mutation associated with an aberrantprotein expression or with a disease condition or state, comprisingdelivering CRISPR-Cas system that targets the target sequence, e.g., viaa delivery system.

For example, the delivering can comprise contacting of one or morenon-naturally occurring or engineered compositions with the HSC orintroducing one or more CRISPR-Cas systems into the HSC. A non-naturallyoccurring or engineered composition can comprise one or more particlescontaining the CRISPR-Cas system or components thereof.

A non-naturally occurring or engineered vector composition can compriseviral, plasmid or nucleic acid molecule vector(s) (e.g. RNA) operablyencoding the CRISPR-Cas composition for expression thereof, wherein thevector composition comprises:

(A) I. a first regulatory element operably linked to a CRISPR-Cas systemchimeric RNA (chiRNA) polynucleotide sequence, wherein thepolynucleotide sequence comprises (a) a guide sequence capable ofhybridizing to a target sequence in a eukaryotic cell, (b) a tracr matesequence, and (c) a tracr sequence, and II. a second regulatory elementoperably linked to an enzyme-coding sequence encoding a CRISPR enzymecomprising at least one or more nuclear localization sequences (oroptionally at least one or more nuclear localization sequences as someembodiments can involve no NLS), wherein (a), (b) and (c) are arrangedin a 5′ to 3′ orientation, wherein components I and II are located onthe same or different vectors of the system, wherein when transcribed,the tracr mate sequence hybridizes to the tracr sequence and the guidesequence directs sequence-specific binding of a CRISPR complex to thetarget sequence, and wherein the CRISPR complex comprises the CRISPRenzyme complexed with (1) the guide sequence that is hybridized to thetarget sequence, and (2) the tracr mate sequence that is hybridized tothe tracr sequence, or (B) a non-naturally occurring or engineeredcomposition comprising a vector system comprising one or more vectorscomprising I. a first regulatory element operably linked to (a) a guidesequence capable of hybridizing to a target sequence in a eukaryoticcell, and (b) at least one or more tracr mate sequences, II. a secondregulatory element operably linked to an enzyme-coding sequence encodinga CRISPR enzyme, and III. a third regulatory element operably linked toa tracr sequence, wherein components I, II and III are located on thesame or different vectors of the system, wherein when transcribed, thetracr mate sequence hybridizes to the tracr sequence and the guidesequence directs sequence-specific binding of a CRISPR complex to thetarget sequence, and wherein the CRISPR complex comprises the CRISPRenzyme complexed with (1) the guide sequence that is hybridized to thetarget sequence, and (2) the tracr mate sequence that is hybridized tothe tracr sequence; the method may optionally include also delivering aHDR template, e.g., via the particle contacting the HSC containing orcontacting the HSC with another particle containing, the HDR templatewherein the HDR template provides expression of a normal or lessaberrant form of the protein; wherein “normal” is as to wild type, and“aberrant” can be a protein expression that gives rise to a condition ordisease state; and optionally the method may include isolating orobtaining HSC from the organism or non-human organism, optionallyexpanding the HSC population, performing contacting of the particle(s)with the HSC to obtain a modified HSC population, optionally expandingthe population of modified HSCs, and optionally administering modifiedHSCs to the organism or non-human organism. In some embodiments,components I, II and III are located on the same vector. In otherembodiments, components I and II are located on the same vector, whilecomponent III is located on another vector. In other embodiments,components I and III are located on the same vector, while component IIis located on another vector. In other embodiments, components II andIII are located on the same vector, while component I is located onanother vector. In other embodiments, each of components I, II and IIIis located on different vectors. The invention also provides a viral orplasmid vector system as described herein.

By manipulation of a target sequence, Applicants also mean theepigenetic manipulation of a target sequence. This may be of thechromatin state of a target sequence, such as by modification of themethylation state of the target sequence (i.e. addition or removal ofmethylation or methylation patterns or CpG islands), histonemodification, increasing or reducing accessibility to the targetsequence, or by promoting 3D folding. It will be appreciated that wherereference is made to a method of modifying an organism or mammalincluding human or a non-human mammal or organism by manipulation of atarget sequence in a locus, e.g., genomic locus of interest, this mayapply to the organism (or mammal) as a whole or just a single cell orpopulation of cells from that organism (if the organism ismulticellular). In the case of humans, for instance, Applicantsenvisage, inter alia, a single cell or a population of cells and thesemay preferably be modified ex vivo and then introduced or re-introduced.In this case, a biopsy or other tissue or biological fluid sample may benecessary. Stem cells are also particularly preferred in this regard.But, of course, in vivo embodiments are also envisaged. And theinvention is especially advantageous as to HSCs.

The invention in some embodiments comprehends a method of modifying anorganism or a non-human organism by manipulation of a first and a secondtarget sequence on opposite strands of a polynucleotide, e.g., DNA,duplex, for instance, in a locus, e.g., genomic locus of interest, in aHSC e.g., wherein the locus or genomic locus of interest is associatedwith a mutation associated with an aberrant protein expression or with adisease condition or state, comprising delivering, e.g., by contactingHSCs with particle(s) comprising a non-naturally occurring or engineeredcomposition comprising:

-   -   I. a first CRISPR-Cas system chimeric RNA (chiRNA)        polynucleotide sequence, wherein the first polynucleotide        sequence comprises:        -   (a) a first guide sequence capable of hybridizing to the            first target sequence,        -   (b) a first tracr mate sequence, and        -   (c) a first tracr sequence,    -   II. a second CRISPR-Cas system chiRNA polynucleotide sequence,        wherein the second polynucleotide sequence comprises:        -   (a) a second guide sequence capable of hybridizing to the            second target sequence,        -   (b) a second tracr mate sequence, and        -   (c) a second tracr sequence, and    -   III. a polynucleotide sequence encoding a CRISPR enzyme        comprising at least one or more nuclear localization sequences        and comprising one or more mutations, wherein (a), (b) and (c)        are arranged in a 5′ to 3′ orientation; or    -   IV. expression product(s) of one or more of I. to III., e.g.,        the the first and the second tracr mate sequence, the CRISPR        enzyme;

wherein when transcribed, the first and the second tracr mate sequencehybridize to the first and second tracr sequence respectively and thefirst and the second guide sequence directs sequence-specific binding ofa first and a second CRISPR complex to the first and second targetsequences respectively, wherein the first CRISPR complex comprises theCRISPR enzyme complexed with (1) the first guide sequence that ishybridized to the first target sequence, and (2) the first tracr matesequence that is hybridized to the first tracr sequence, wherein thesecond CRISPR complex comprises the CRISPR enzyme complexed with (1) thesecond guide sequence that is hybridized to the second target sequence,and (2) the second tracr mate sequence that is hybridized to the secondtracr sequence, wherein the polynucleotide sequence encoding a CRISPRenzyme is DNA or RNA, and wherein the first guide sequence directscleavage of one strand of the DNA duplex near the first target sequenceand the second guide sequence directs cleavage of the other strand nearthe second target sequence inducing a double strand break, therebymodifying the organism or the non-human organism; and the method mayoptionally include also delivering a HDR template, e.g., via theparticle contacting the HSC containing or contacting the HSC withanother particle containing, the HDR template wherein the HDR templateprovides expression of a normal or less aberrant form of the protein;wherein “normal” is as to wild type, and “aberrant” can be a proteinexpression that gives rise to a condition or disease state; andoptionally the method may include isolating or obtaining HSC from theorganism or non-human organism, optionally expanding the HSC population,performing contacting of the particle(s) with the HSC to obtain amodified HSC population, optionally expanding the population of modifiedHSCs, and optionally administering modified HSCs to the organism ornon-human organism. In some methods of the invention any or all of thepolynucleotide sequence encoding the CRISPR enzyme, the first and thesecond guide sequence, the first and the second tracr mate sequence orthe first and the second tracr sequence, is/are RNA. In furtherembodiments of the invention the polynucleotides encoding the sequenceencoding the CRISPR enzyme, the first and the second guide sequence, thefirst and the second tracr mate sequence or the first and the secondtracr sequence, is/are RNA and are delivered via liposomes,nanoparticles, exosomes, microvesicles, or a gene-gun; but, it isadvantageous that the delivery is via a particle. In certain embodimentsof the invention, the first and second tracr mate sequence share 100%identity and/or the first and second tracr sequence share 100% identity.In some embodiments, the polynucleotides may be comprised within avector system comprising one or more vectors. In preferred embodimentsof the invention the CRISPR enzyme is a Cas9 enzyme, e.g. SpCas9. In anaspect of the invention the CRISPR enzyme comprises one or moremutations in a catalytic domain, wherein the one or more mutations, withreference to SpCas9 are selected from the group consisting of D10A,E762A, H840A, N854A, N863A and D986A, e.g., a D10A mutation. Inpreferred embodiments, the first CRISPR enzyme has one or more mutationssuch that the enzyme is a complementary strand nicking enzyme, and thesecond CRISPR enzyme has one or more mutations such that the enzyme is anon-complementary strand nicking enzyme. Alternatively the first enzymemay be a non-complementary strand nicking enzyme, and the second enzymemay be a complementary strand nicking enzyme. In preferred methods ofthe invention the first guide sequence directing cleavage of one strandof the DNA duplex near the first target sequence and the second guidesequence directing cleavage of the other strand near the second targetsequence results in a 5′ overhang. In embodiments of the invention the5′ overhang is at most 200 base pairs, preferably at most 100 basepairs, or more preferably at most 50 base pairs. In embodiments of theinvention the 5′ overhang is at least 26 base pairs, preferably at least30 base pairs or more preferably 34-50 base pairs.

The invention in some embodiments comprehends a method of modifying anorganism or a non-human organism by manipulation of a first and a secondtarget sequence on opposite strands of a DNA duplex in a genomic locusof interest in a HSC e.g., wherein the genomic locus of interest isassociated with a mutation associated with an aberrant proteinexpression or with a disease condition or state, comprising delivering,e.g., by contacting HSCs with particle(s) comprising a non-naturallyoccurring or engineered composition comprising:

-   -   I. a first regulatory element operably linked to        -   (a) a first guide sequence capable of hybridizing to the            first target sequence, and        -   (b) at least one or more tracr mate sequences,    -   II. a second regulatory element operably linked to        -   (a) a second guide sequence capable of hybridizing to the            second target sequence, and        -   (b) at least one or more tracr mate sequences,    -   III. a third regulatory element operably linked to an        enzyme-coding sequence encoding a CRISPR enzyme, and    -   IV. a fourth regulatory element operably linked to a tracr        sequence,    -   V. expression product(s) of one or more of I. to IV., e.g., the        the first and the second tracr mate sequence, the CRISPR enzyme;

Components I, II, III and IV can be located on the same or differentvectors and/or be contained in same or different particles of thesystem; advantageously they are in the same vector and/or same particle.

When transcribed, the first and the second guide sequence directsequence-specific binding of a first and a second CRISPR complex to thefirst and second target sequences respectively. If present in andapplicable to the CRISPR-Cas system, the tracr mate sequence hybridizesto the tracr sequence. A first CRISPR complex comprises the CRISPRenzyme complexed with the first guide sequence that is hybridized to thefirst target sequence; and optionally if present in and applicable tothe CRISPR-Cas system, the tracr mate sequence that is hybridized to thetracr sequence. A second CRISPR complex comprises the CRISPR enzymecomplexed with the second guide sequence that is hybridized to thesecond target sequence; and optionally if present in and applicable tothe CRISPR-Cas system, the tracr mate sequence that is hybridized to thetracr sequence. The polynucleotide sequence encoding the CRISPR enzymecan be DNA or RNA. The first guide sequence directs cleavage of onestrand of the polynucleotide, e.g., DNA, duplex near the first targetsequence. The second guide sequence directs cleavage of the other strandnear the second target sequence inducing a double strand break;advantageously the breaks have overhangs.

The method may optionally include also delivering a polynucleotide,e.g., HDR, template, e.g., via a delivery system used to deliverCRISPR-Cas system(s) or component(s) thereof, e.g., the particlecontacting the HSC containing; or contacting the HSC with anotherparticle containing, the template. In instances where a complexed orassembled CRISPR-Cas system is delivered via a particle, the particlecan include the template, e.g., hybridization bonded or pre-annealed toRNA of the system. The template provides expression of a normal or lessaberrant form of the protein; wherein “normal” is as to wild type, and“aberrant” can be a protein expression that gives rise to a condition ordisease state. The template can be DNA or RNA, depending upon theCRISPR-Cas system enzyme or protein. Optionally the method may includeisolating or obtaining HSC from the organism or non-human organism. Themethod may also optionally include expanding the HSC population, e.g.,performing a herein method to obtain a modified HSC population, andexpanding the population of modified HSCs. The method can optionallyinclude administering modified HSCs to the organism or non-humanorganism. HSCs can be obtained from a first organism or non-humanorganism, subjected to a herein method to obtain a modified HSCpopulation, the modified HSC population may optionally be expanded, andmodified HSCs can be administered to a second organism or non-humanorganism. In a personalized medicine therapy, the first and secondorganism or non-human organism can be the same. In other instances, thefirst and second organisms can be sufficiently related that cells fromthe first can be administered to the second (e.g., cells from abiological relative, a compatible donor)

The invention also provides a vector system as described herein. Thesystem may comprise one, two, three or four different vectors.Components I, II, III and IV may thus be located on one, two, three orfour different vectors, and all combinations for possible locations ofthe components are herein envisaged, for example: components I, II, IIIand IV can be located on the same vector; components I, II, III and IVcan each be located on different vectors; components I, II, III and IVmay be located on a total of two or three different vectors, with allcombinations of locations envisaged, etc. In some methods of theinvention any or all of the polynucleotide sequence encoding the CRISPRenzyme, the first and the second guide sequence, the first and thesecond tracr mate sequence if applicable to the CRISPR-Cas system or thefirst and the second tracr sequence if applicable to the CRISPR-Cassystem, is/are RNA. In further embodiments of the invention the firstand second tracr mate sequence if applicable to the CRISPR-Cas systemshare 100% identity and/or the first and second tracr sequence ifapplicable to the CRISPR-Cas system share 100% identity. In certainpreferred embodiments of the invention the CRISPR enzyme is a Cas9enzyme, e.g. SpCas9 or SaCas9. In an aspect of the invention the CRISPRenzyme comprises one or more mutations in a catalytic domain, whereinthe one or more mutations with reference to SpCas9 are selected from thegroup consisting of D10A, E762A, H840A, N854A, N863A and D986A; e.g.,D10A mutation. In preferred embodiments, the first CRISPR enzyme has oneor more mutations such that the enzyme is a complementary strand nickingenzyme, and the second CRISPR enzyme has one or more mutations such thatthe enzyme is a non-complementary strand nicking enzyme. Alternativelythe first enzyme may be a non-complementary strand nicking enzyme, andthe second enzyme may be a complementary strand nicking enzyme. In afurther embodiment of the invention, one or more of the viral vectorsare delivered via liposomes, nanoparticles, exosomes, microvesicles, ora gene-gun; but, particle delivery is advantageous.

In preferred methods of the invention the first guide sequence directingcleavage of one strand of the DNA duplex near the first target sequenceand the second guide sequence directing cleavage of other strand nearthe second target sequence results in a 5′ overhang. In embodiments ofthe invention the 5′ overhang is at most 200 base pairs, preferably atmost 100 base pairs, or more preferably at most 50 base pairs. Inembodiments of the invention the 5′ overhang is at least 26 base pairs,preferably at least 30 base pairs or more preferably 34-50 base pairs.

The invention in some embodiments comprehends a method of modifying agenomic locus of interest in HSC e.g., wherein the genomic locus ofinterest is associated with a mutation associated with an aberrantprotein expression or with a disease condition or state, by introducinginto the HSC, e.g., by contacting HSCs with particle(s) comprising, aCas protein having one or more mutations and two guide RNAs that targeta first strand and a second strand of the DNA molecule respectively inthe HSC, whereby the guide RNAs target the DNA molecule and the Casprotein nicks each of the first strand and the second strand of the DNAmolecule, whereby a target in the HSC is altered; and, wherein the Casprotein and the two guide RNAs do not naturally occur together and themethod may optionally include also delivering a HDR template, e.g., viathe particle contacting the HSC containing or contacting the HSC withanother particle containing, the HDR template wherein the HDR templateprovides expression of a normal or less aberrant form of the protein;wherein “normal” is as to wild type, and “aberrant” can be a proteinexpression that gives rise to a condition or disease state; andoptionally the method may include isolating or obtaining HSC from theorganism or non-human organism, optionally expanding the HSC population,performing contacting of the particle(s) with the HSC to obtain amodified HSC population, optionally expanding the population of modifiedHSCs, and optionally administering modified HSCs to the organism ornon-human organism. In preferred methods of the invention the Casprotein nicking each of the first strand and the second strand of theDNA molecule results in a 5′ overhang. In embodiments of the inventionthe 5′ overhang is at most 200 base pairs, preferably at most 100 basepairs, or more preferably at most 50 base pairs. In embodiments of theinvention the 5′ overhang is at least 26 base pairs, preferably at least30 base pairs or more preferably 34-50 base pairs. Embodiments of theinvention also comprehend the guide RNAs comprising a guide sequencefused to a tracr mate sequence and a tracr sequence. In an aspect of theinvention the Cas protein is codon optimized for expression in aeukaryotic cell, preferably a mammalian cell or a human cell. In furtherembodiments of the invention the Cas protein is a type II CRISPR-Casprotein, e.g. a Cas 9 protein. In a highly preferred embodiment the Casprotein is a Cas9 protein, e.g. SpCas9 or SaCas9. In aspects of theinvention the Cas protein has one or more mutations in respect of SpCas9selected from the group consisting of D10A, E762A, H840A, N854A, N863Aand D986A; e.g., a D10A mutation. Aspects of the invention relate to theexpression of a gene product being decreased or a templatepolynucleotide being further introduced into the DNA molecule encodingthe gene product or an intervening sequence being excised precisely byallowing the two 5′ overhangs to reanneal and ligate or the activity orfunction of the gene product being altered or the expression of the geneproduct being increased. In an embodiment of the invention, the geneproduct is a protein.

The invention in some embodiments comprehends a method of modifying agenomic locus of interest in HSC e.g., wherein the genomic locus ofinterest is associated with a mutation associated with an aberrantprotein expression or with a disease condition or state, by introducinginto the HSC, e.g., by contacting HSCs with particle(s) comprising,

-   -   a) a first regulatory element operably linked to each of two        CRISPR-Cas system guide RNAs that target a first strand and a        second strand respectively of a double stranded DNA molecule of        the HSC, and    -   b) a second regulatory element operably linked to a Cas protein,        or    -   c) expression product(s) of a) or b),

wherein components (a) and (b) are located on same or different vectorsof the system, whereby the guide RNAs target the DNA molecule of the HSCand the Cas protein nicks each of the first strand and the second strandof the DNA molecule of the HSC; and, wherein the Cas protein and the twoguide RNAs do not naturally occur together; and the method mayoptionally include also delivering a HDR template, e.g., via theparticle contacting the HSC containing or contacting the HSC withanother particle containing, the HDR template wherein the HDR templateprovides expression of a normal or less aberrant form of the protein;wherein “normal” is as to wild type, and “aberrant” can be a proteinexpression that gives rise to a condition or disease state; andoptionally the method may include isolating or obtaining HSC from theorganism or non-human organism, optionally expanding the HSC population,performing contacting of the particle(s) with the HSC to obtain amodified HSC population, optionally expanding the population of modifiedHSCs, and optionally administering modified HSCs to the organism ornon-human organism. In aspects of the invention the guide RNAs maycomprise a guide sequence fused to a tracr mate sequence and a tracrsequence. In an embodiment of the invention the Cas protein is a type IICRISPR-Cas protein. In an aspect of the invention the Cas protein iscodon optimized for expression in a eukaryotic cell, preferably amammalian cell or a human cell. In further embodiments of the inventionthe Cas protein is a type II CRISPR-Cas protein, e.g. a Cas 9 protein.In a highly preferred embodiment the Cas protein is a Cas9 protein, e.g.SpCas9 or SaCas9. In aspects of the invention the Cas protein has one ormore mutations with reference to SpCas9 selected from the groupconsisting of D10A, E762A, H840A, N854A, N863A and D986A; e.g., the D10Amutation. Aspects of the invention relate to the expression of a geneproduct being decreased or a template polynucleotide being furtherintroduced into the DNA molecule encoding the gene product or anintervening sequence being excised precisely by allowing the two 5′overhangs to reanneal and ligate or the activity or function of the geneproduct being altered or the expression of the gene product beingincreased. In an embodiment of the invention, the gene product is aprotein. In preferred embodiments of the invention the vectors of thesystem are viral vectors. In a further embodiment, the vectors of thesystem are delivered via liposomes, nanoparticles, exosomes,microvesicles, or a gene-gun; and particles are preferred. In oneaspect, the invention provides a method of modifying a targetpolynucleotide in a HSC. In some embodiments, the method comprisesallowing a CRISPR complex to bind to the target polynucleotide to effectcleavage of said target polynucleotide thereby modifying the targetpolynucleotide, wherein the CRISPR complex comprises a CRISPR enzymecomplexed with a guide sequence hybridized to a target sequence withinsaid target polynucleotide, wherein said guide sequence is linked to atracr mate sequence which in turn hybridizes to a tracr sequence. Insome embodiments, said cleavage comprises cleaving one or two strands atthe location of the target sequence by said CRISPR enzyme. In someembodiments, said cleavage results in decreased transcription of atarget gene. In some embodiments, the method further comprises repairingsaid cleaved target polynucleotide by homologous recombination with anexogenous template polynucleotide, wherein said repair results in amutation comprising an insertion, deletion, or substitution of one ormore nucleotides of said target polynucleotide. In some embodiments,said mutation results in one or more amino acid changes in a proteinexpressed from a gene comprising the target sequence. In someembodiments, the method further comprises delivering one or more vectorsor expression product(s) thereof, e.g., via particle(s), to said HSC,wherein the one or more vectors drive expression of one or more of: theCRISPR enzyme, the guide sequence linked to the tracr mate sequence, andthe tracr sequence. In some embodiments, said vectors are delivered tothe HSC in a subject. In some embodiments, said modifying takes place insaid HSC in a cell culture. In some embodiments, the method furthercomprises isolating said HSC from a subject prior to said modifying. Insome embodiments, the method further comprises returning said HSC and/orcells derived therefrom to said subject.

In one aspect, the invention provides a method of generating a HSCcomprising a mutated disease gene. In some embodiments, a disease geneis any gene associated with an increase in the risk of having ordeveloping a disease. In some embodiments, the method comprises (a)introducing one or more vectors or expression product(s) thereof, e.g.,via particle(s), into a HSC, wherein the one or more vectors driveexpression of one or more of: a CRISPR enzyme, a guide sequence linkedto a tracr mate sequence, and a tracr sequence; and (b) allowing aCRISPR complex to bind to a target polynucleotide to effect cleavage ofthe target polynucleotide within said disease gene, wherein the CRISPRcomplex comprises the CRISPR enzyme complexed with (1) the guidesequence that is hybridized to the target sequence within the targetpolynucleotide, and (2) the tracr mate sequence that is hybridized tothe tracr sequence, thereby generating a HSC comprising a mutateddisease gene. In some embodiments, said cleavage comprises cleaving oneor two strands at the location of the target sequence by said CRISPRenzyme. In some embodiments, said cleavage results in decreasedtranscription of a target gene. In some embodiments, the method furthercomprises repairing said cleaved target polynucleotide by homologousrecombination with an exogenous template polynucleotide, wherein saidrepair results in a mutation comprising an insertion, deletion, orsubstitution of one or more nucleotides of said target polynucleotide.In some embodiments, said mutation results in one or more amino acidchanges in a protein expression from a gene comprising the targetsequence. In some embodiments the modified HSC is administered to ananimal to thereby generate an animal model.

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a HSC. In some embodiments, the method comprisesallowing a CRISPR complex to bind to the target polynucleotide to effectcleavage of said target polynucleotide thereby modifying the targetpolynucleotide, wherein the CRISPR complex comprises a CRISPR enzymecomplexed with a guide sequence hybridized to a target sequence withinsaid target polynucleotide, wherein said guide sequence is linked to atracr mate sequence which in turn hybridizes to a tracr sequence. Inother embodiments, this invention provides a method of modifyingexpression of a polynucleotide in a eukaryotic cell that arises from anHSC. The method comprises increasing or decreasing expression of atarget polynucleotide by using a CRISPR complex that binds to thepolynucleotide in the HSC; advantageously the CRISPR complex isdelivered via particle(s).

In some methods, a target polynucleotide can be inactivated to effectthe modification of the expression in a HSC. For example, upon thebinding of a CRISPR complex to a target sequence in a cell, the targetpolynucleotide is inactivated such that the sequence is not transcribed,the coded protein is not produced, or the sequence does not function asthe wild-type sequence does.

In some embodiments the RNA of the CRISPR-Cas system, e.g., the guide orsgRNA, can be modified; for instance to include an aptamer or afunctional domain. An aptamer is a synthetic oligonucleotide that bindsto a specific target molecule; for instance a nucleic acid molecule thathas been engineered through repeated rounds of in vitro selection orSELEX (systematic evolution of ligands by exponential enrichment) tobind to various molecular targets such as small molecules, proteins,nucleic acids, and even cells, tissues and organisms. Aptamers areuseful in that they offer molecular recognition properties that rivalthat of antibodies. In addition to their discriminate recognition,aptamers offer advantages over antibodies including that they elicitlittle or no immunogenicity in therapeutic applications. Accordingly, inthe practice of the invention, either or both of the enzyme or the RNAcan include a functional domain.

In some embodiments, the functional domain is a transcriptionalactivation domain, preferably VP64. In some embodiments, the functionaldomain is a transcription repression domain, preferably KRAB. In someembodiments, the transcription repression domain is SID, or concatemersof SID (eg SID4X). In some embodiments, the functional domain is anepigenetic modifying domain, such that an epigenetic modifying enzyme isprovided. In some embodiments, the functional domain is an activationdomain, which may be the P65 activation domain.

The invention further comprehends a composition of the invention or aCRISPR complex or enzyme thereof or RNA thereof (including oralternatively mRNA encoding the CRISPR enzyme) for use in medicine or intherapy. In some embodiments the invention comprehends a compositionaccording to the invention or components thereof for use in a methodaccording to the invention. In some embodiments the invention providesfor the use of a composition of the invention or a CRISPR complex orenzyme thereof or RNA thereof (including or alternatively mRNA encodingthe CRISPR enzyme) in ex vivo gene or genome editing, especially in HSCswhich optionally may then be introduced into an organism or non-humanorganism from which the HSCs were obtained or another organism ornon-human organism of the same species. In certain embodiments theinvention comprehends use of a composition of the invention or a CRISPRcomplex or enzyme thereof or RNA thereof (including or alternativelymRNA encoding the CRISPR enzyme) in the manufacture of a medicament forex vivo gene or genome editing or for use in a method according of theinvention. In certain embodiments the invention provides a method oftreating or inhibiting a condition caused by a defect in a targetsequence in a genomic locus of interest in a subject (e.g., mammal orhuman) or a non-human subject (e.g., mammal) in need thereof comprisingmodifying HSCs of the subject or a non-human subject by manipulation ofthe target sequence in the HSC and administering the modified HSCs tothe subject or non-human subject, advantageously the modifying of theHSCs is through contacting the HSCs with a particle containing theCRISPR complex or the components thereof, advantageously in certainembodiments the particle also provides a HDR template or anotherparticle or a vector provides the HDR template, and wherein thecondition is susceptible to treatment or inhibition by manipulation ofthe target sequence.

Certain RNA of the CRISPR Cas complex is also known and referred to assgRNA (single guide RNA). In advantageous embodiments RNA of the CRISPRCas complex is sgRNA. The CRISPR-Cas9 system has been engineered totarget genetic locus or loci in HSCs. Cas9 protein, advantageouslycodon-optimized for a eukaryotic cell and especially a mammalian cell,e.g., a human cell, for instance, HSC, and sgRNA targeting a locus orloci in HSC, e.g., the gene EMX1, were prepared. These wereadvantageously delivered via particles. The particles were formed by theCas9 protein and the sgRNA being admixed. The sgRNA and Cas9 proteinmixture was admixed with a mixture comprising or consisting essentiallyof or consisting of surfactant, phospholipid, biodegradable polymer,lipoprotein and alcohol, whereby particles containing the sgRNA and Cas9protein were formed. The invention comprehends so making particles andparticles from such a method as well as uses thereof. More generally,particles were formed using an efficient process. First, Cas9 proteinand sgRNA targeting the gene EMX1 or the control gene LacZ were mixedtogether at a suitable, e.g., 3:1 to 1:3 or 2:1 to 1:2 or 1:1 molarratio, at a suitable temperature, e.g., 15-30 C, e.g., 20-25 C, e.g.,room temperature, for a suitable time, e.g., 15-45, such as 30 minutes,advantageously in sterile, nuclease free buffer, e.g., 1×PBS.Separately, particle components such as or comprising: a surfactant,e.g., cationic lipid, e.g., 1,2-dioleoyl-3-trimethylammonium-propane(DOTAP); phospholipid, e.g., dimyristoylphosphatidylcholine (DMPC);biodegradable polymer, such as an ethylene-glycol polymer or PEG, and alipoprotein, such as a low-density lipoprotein, e.g., cholesterol weredissolved in an alcohol, advantageously a C₁₋₆ alkyl alcohol, such asmethanol, ethanol, isopropanol, e.g., 100% ethanol. The two solutionswere mixed together to form particles containing the Cas9-sgRNAcomplexes. In certain embodiments the particle can contain an HDRtemplate. That can be a particle co-administered with sgRNA+Cas9protein-containing particle, or i.e., in addition to contacting an HSCwith an sgRNA+Cas9 protein-containing particle, the HSC is contactedwith a particle containing an HDR template; or the HSC is contacted witha particle containing all of the sgRNA, Cas9 and the HDR template. TheHDR template can be administered by a separate vector, whereby in afirst instance the particle penetrates an HSC cell and the separatevector also penetrates the cell, wherein the HSC genome is modified bythe sgRNA+Cas9 and the HDR template is also present, whereby a genomicloci is modified by the HDR; for instance, this may result in correctinga mutation. The particle in the herein discussion is advantageouslyobtained or obtainable from admixing an sgRNA(s) and Cas9 proteinmixture (optionally containing HDR template(s) or such mixture onlycontaining HDR template(s) when separate particles as to template(s) isdesired) with a mixture comprising or consisting essentially of orconsisting of surfactant, phospholipid, biodegradable polymer,lipoprotein and alcohol (wherein one or more sgRNA targets the geneticlocus or loci in the HSC).

In herein discussions concerning the target being associated with amutation or with a disease condition, such mutation or disease conditioncan be, for instance Hemophilia B, SCID, SCID-X1, ADA-SCID, Hereditarytyrosinemia, β-thalassemia, X-linked CGD, Wiskott-Aldrich syndrome,Fanconi anemia, adrenoleukodystrophy (ALD), metachromatic leukodystrophy(MLD), or HIV/AIDS; or more generally an Immunodeficiency disorder,Hematologic condition, or genetic lysosomal storage disease.

Hematopoietic stem cells treatments are also relied on to alleviatecertain aspects of genetic disorders. Alpha-mannosidosis is inherited inan autosomal recessive fashion and is caused by a spectrum of mutationsin the MAN2B1 gene located on chromosome 19 (19 p13.2-q12). One study of39 families identified eight splicing, six missense, and three nonsensemutations, as well as two small insertions and two small deletions (seeBerg et al., January 1999, Am. J. Hum. Genet. 64(1): 77-88. Grewall etal., (J Pediatr. 2004 May; 144(5):569-73) treated alpha-mannosidosis bytransplanting allogeneic hematopoietic stem cell. Alpha-mannosidosis canbe treated by transplantation of a subjects own hematopoietic stem cellsprepared according to the invention to correct deficient expression ofMAN2B1.

Leukodystrophies are a group on inherited diseases in which molecularabnormalities of glial cells are responsible defects in myelin formationand/or maintenance within the central and peripheral nervous system. Ingloboid cell leukodystrophy (Krabbe Disease) there is an inheritedmetabolic disorder of the central nervous system caused by deficiency ofthe lysosomal enzyme galactocerebrosidase (GALC). The mutationalspectrum comprises missense mutations in evolutionarily conservedresidues as well as frameshift mutations, nonsense mutations, andmutations that mediate exon skipping. Hematopoietic stem celltransplantation has provided an effective treatment. The engraftmentfrom normal donors provides cells able to correct the metabolic defectsee Tappino et al., December 2010, Hum Mutat. 31(12):E1894-914. doi:10.1002/humu.21367. Leukodistrophies can be treated by transplantationof a subjects own hematopoietic stem cells prepared according to theinvention.

Polycythemia vera (PCV), being is caused by neoplastic proliferation oferythroid, megakaryocytic and granulocytic cells. PCV is associated witha mutation in the tyrosine kinaseJAK2 (usually V617F), which acts insignaling pathways of the EPO-receptor, rendering those cellshypersensitive to EPO. (See, Lussana et al., May 2014, Haematologica,99(5):916-21. doi: 10.3324/haematol.2013.094284. Epub 2014 Jan. 3).Allogeneic stem cell transplantation is currently the only potentiallycurative treatment. PCV can be treated by transplantation of a subjectsown hematopoietic stem cells prepared according to the invention.Calreticulin (CALR) mutations are the second most common cause ofmyeloproliferative neoplasms. Consequences in PCV include double thenormal number of erythrocytes per cubic millimeter of blood, elevatedhematocrit, and twice normal blood volume. Familial essentialthrombocythaemia (ET) is another congenital disease inherited in anautosomal dominant manner. ET is characterized by overproduction ofplatelets by megakaryocytes in the bone marrow, and alterations of theTPO receptor encoded for by the c-mpl gene are associated. According tothe invention, HSCs obtained from subjects afflicted with suchmyeloproliferative disorders are subject to gene editing to correct thecausal mutation. The patient's own gene edited stem cells may then beadministered to the patient.

As to a CRISPR-Cas system of the invention being engineered ornon-naturally occurring, the RNA of the system can be engineered, e.g.,to target a nucleic acid molecule in an HSC and/or the protein and/orthe nucleic acid molecule encoding it can be engineered, e.g., codonoptimized for expression in a HSC and/or a having a functionalizedeffector, e.g., NLS, Fok I, etc and/or having one or more a mutation(s)or truncation(s), e.g., as to having the protein become a nicking enzymeor an enzyme that binds without significant cutting (e.g., less than 5%cutting activity) and/or wherein the protein is a chimeric of two ormore CRISPR-Cas proteins.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention. It may be advantageous in the practiceof the invention to be in compliance with Art. 53(c) EPC and Rule 28(b)and (c) EPC. Nothing herein is to be construed as a promise.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows comparison of different programmable nuclease platforms.

FIG. 2A-2C shows Types of Therapeutic Genome Modifications. The specifictype of genome editing therapy depends on the nature of the mutationcausing disease. a, In gene disruption, the pathogenic function of aprotein is silenced by targeting the locus with NHEJ. Formation ofindels on the gene of interest often result in frameshift mutations thatcreate premature stop codons and a non-functional protein product, ornon-sense mediated decay of transcripts, suppressing gene function. b,HDR gene correction can be used to correct a deleterious mutation. DSBis targeted near the mutation site in the presence of an exogenouslyprovided, corrective HDR template. HDR repair of the break site with theexogenous template corrects the mutation, restoring gene function. c, Analternative to gene correction is gene addition. This mode of treatmentintroduces a therapeutic transgene into a safe-harbor locus in thegenome. DSB is targeted to the safe-harbor locus and an HDR templatecontaining homology to the break site, a promoter and a transgene isintroduced to the nucleus. HDR repair copies the promoter-transgenecassette into the safe-harbor locus, recovering gene function, albeitwithout true physiological control over gene expression.

FIG. 3 shows Ex vivo vs. in vivo editing therapy. In ex vivo editingtherapy cells are removed from a patient, edited and then re-engrafted(top panel). For this mode of therapy to be successful, target cellsmust be capable of survival outside the body and homing back to targettissues post-transplantation. In vivo therapy involves genome editing ofcells in situ (bottom panels). For in vivo systemic therapy, deliveryagents that are relatively agnostic to cell identity or state would beused to effect editing in a wide range of tissue types. Although thismode of editing therapy may be possible in the future, no deliverysystems currently exist that are efficient enough to make this feasible.In vivo targeted therapy, where delivery agents with tropism forspecific organ systems are administered to patients are feasible withclinically relevant viral vectors.

FIG. 4 shows a schematic representation of gene therapy via Cas9Homologous Recombination (HR) vectors.

The figures herein are for illustrative purposes only and are notnecessarily drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

With respect to general information on CRISPR-Cas Systems, componentsthereof, and delivery of such components, including methods, materials,delivery vehicles, vectors, particles, AAV, and making and usingthereof, including as to amounts and formulations, all useful in thepractice of the instant invention, reference is made to: U.S. Pat. Nos.8,697,359, 8,771,945, 8,795,965, 8,865,406, 8,871,445, 8,889,356,8,889,418 and 8,895,308; US Patent Publications US 2014-0310830 (U.S.application Ser. No. 14/105,031), US 2014-0287938 A1 (U.S. applicationSer. No. 14/213,991), US 2014-0273234 A1 (U.S. application Ser. No.14/293,674), US2014-0273232 A1 (U.S. application Ser. No. 14/290,575),US 2014-0273231 (U.S. application Ser. No. 14/259,420), US 2014-0256046A1 (U.S. application Ser. No. 14/226,274), US 2014-0248702 A1 (U.S.application Ser. No. 14/258,458), US 2014-0242700 A1 (U.S. applicationSer. No. 14/222,930), US 2014-0242699 A1 (U.S. application Ser. No.14/183,512), US 2014-0242664 A1 (U.S. application Ser. No. 14/104,990),US 2014-0234972 A1 (U.S. application Ser. No. 14/183,471), US2014-0227787 A1 (U.S. application Ser. No. 14/256,912), US 2014-0189896A1 (U.S. application Ser. No. 14/105,035), US 2014-0186958 (U.S.application Ser. No. 14/105,017), US 2014-0186919 A1 (U.S. applicationSer. No. 14/104,977), US 2014-0186843 A1 (U.S. application Ser. No.14/104,900), US 2014-0179770 A1 (U.S. application Ser. No. 14/104,837)and US 2014-0179006 A1 (U.S. application Ser. No. 14/183,486), US2014-0170753 (U.S. application Ser. No. 14/183,429); European PatentApplications EP 2 771 468 (EP13818570.7), EP 2 764 103 (EP13824232.6),and EP 2 784 162 (EP14170383.5); and PCT Patent Publications WO2014/093661 (PCT/US2013/074743), WO 2014/093694 (PCT/US2013/074790), WO2014/093595 (PCT/US2013/074611), WO 2014/093718 (PCT/US2013/074825), WO2014/093709 (PCT/US2013/074812), WO 2014/093622 (PCT/US2013/074667), WO2014/093635 (PCT/US2013/074691), WO 2014/093655 (PCT/US2013/074736), WO2014/093712 (PCT/US2013/074819), WO 2014/093701 (PCT/US2013/074800), WO2014/018423 (PCT/US2013/051418), WO 2014/204723 (PCT/US2014/041790), WO2014/204724 (PCT/US2014/041800), WO 2014/204725 (PCT/US2014/041803), WO2014/204726 (PCT/US2014/041804), WO 2014/204727 (PCT/US2014/041806), WO2014/204728 (PCT/US2014/041808), and WO 2014/204729 (PCT/US2014/041809).Reference is also made to U.S. provisional patent applications61/758,468; 61/802,174; 61/806,375; 61/814,263; 61/819,803 and61/828,130, filed on Jan. 30, 2013; Mar. 15, 2013; Mar. 28, 2013; Apr.20, 2013; May 6, 2013 and May 28, 2013 respectively. Reference is alsomade to U.S. provisional patent application 61/836,123, filed on Jun.17, 2013. Reference is additionally made to U.S. provisional patentapplications 61/835,931, 61/835,936, 61/836,127, 61/836,101, 61/836,080and 61/835,973, each filed Jun. 17, 2013. Further reference is made toU.S. provisional patent applications 61/862,468 and 61/862,355 filed onAug. 5, 2013; 61/871,301 filed on Aug. 28, 2013; 61/960,777 filed onSep. 25, 2013 and 61/961,980 filed on Oct. 28, 2013. Reference is yetfurther made to: PCT Patent applications Nos: PCT/US2014/041803,PCT/US2014/041800, PCT/US2014/041809, PCT/US2014/041804 andPCT/US2014/041806, each filed Jun. 10, 2014 6/10/14; PCT/US2014/041808filed Jun. 11, 2014; and PCT/US2014/62558 filed Oct. 28, 2014, and U.S.Provisional Patent Applications Ser. Nos. 61/915,150, 61/915,301,61/915,267 and 61/915,260, each filed Dec. 12, 2013; 61/757,972 and61/768,959, filed on Jan. 29, 2013 and Feb. 25, 2013; 61/835,936,61/836,127, 61/836,101, 61/836,080, 61/835,973, and 61/835,931, filedJun. 17, 2013; 62/010,888 and 62/010,879, both filed Jun. 11, 2014;62/010,329 and 62/010,441, each filed Jun. 10, 2014; 61/939,228 and61/939,242, each filed Feb. 12, 2014; 61/980,012, filed Apr. 15,2014;62/038,358, filed Aug. 17, 2014; 62/054,490, 62/055,484, 62/055,460 and62/055,487, each filed Sep. 25, 2014; and 62/069,243, filed Oct. 27,2014. Reference is also made to U.S. provisional patent applicationsNos. 62/055,484, 62/055,460, and 62/055,487, filed Sep. 25, 2014; U.S.provisional patent application 61/980,012, filed Apr. 15, 2014; and U.S.provisional patent application 61/939,242 filed Feb. 12, 2014. Referenceis made to PCT application designating, inter alia, the United States,application No. PCT/US14/41806, filed Jun. 10, 2014. Reference is madeto U.S. provisional patent application 61/930,214 filed on Jan. 22,2014. Reference is made to U.S. provisional patent applications61/915,251; 61/915,260 and 61/915,267, each filed on Dec. 12, 2013.Reference is made to US provisional patent application U.S. Ser. No.61/980,012 filed Apr. 15, 2014. Reference is made to PCT applicationdesignating, inter alia, the United States, application No.PCT/US14/41806, filed Jun. 10, 2014. Reference is made to U.S.provisional patent application 61/930,214 filed on Jan. 22, 2014.Reference is made to U.S. provisional patent applications 61/915,251;61/915,260 and 61/915,267, each filed on Dec. 12, 2013.

Mention is also made of U.S. application 62/091,455, filed, 12 Dec. 14,PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/096,708, 24 Dec. 14,PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/091,462, 12 Dec. 14,DEAD GUIDES FOR CRISPR TRANSCRIPTION FACTORS; U.S. application62/096,324, 23 Dec. 14, DEAD GUIDES FOR CRISPR TRANSCRIPTION FACTORS;U.S. application 62/091,456, 12 Dec. 14, ESCORTED AND FUNCTIONALIZEDGUIDES FOR CRISPR-CAS SYSTEMS; U.S. application 62/091,461, 12 Dec. 14,DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS ANDCOMPOSITIONS FOR GENOME EDITING AS TO HEMATOPOETIC STEM CELLS (HSCs);U.S. application 62/094,903, 19 Dec. 14, UNBIASED IDENTIFICATION OFDOUBLE-STRAND BREAKS AND GENOMIC REARRANGEMENT BY GENOME-WISE INSERTCAPTURE SEQUENCING; U.S. application 62/096,761, 24 Dec. 14, ENGINEERINGOF SYSTEMS, METHODS AND OPTIMIZED ENZYME AND GUIDE SCAFFOLDS FORSEQUENCE MANIPULATION; U.S. application 62/098,059, 30 Dec. 14,RNA-TARGETING SYSTEM; U.S. application 62/096,656, 24 Dec. 14, CRISPRHAVING OR ASSOCIATED WITH DESTABILIZATION DOMAINS; U.S. application62/096,697, 24 Dec. 14, CRISPR HAVING OR ASSOCIATED WITH AAV; U.S.application 62/098,158, 30 Dec. 14, ENGINEERED CRISPR COMPLEXINSERTIONAL TARGETING SYSTEMS; U.S. application 62/151,052, 22 Apr. 15,CELLULAR TARGETING FOR EXTRACELLULAR EXOSOMAL REPORTING; U.S.application 62/054,490, 24 Sep. 14, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETINGDISORDERS AND DISEASES USING PARTICLE DELIVERY COMPONENTS; U.S.application 62/055,484, 25 Sep. 14, SYSTEMS, METHODS AND COMPOSITIONSFOR SEQUENCE MANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS;U.S. application 62/087,537, 4 Dec. 14, SYSTEMS, METHODS ANDCOMPOSITIONS FOR SEQUENCE MANIPULATION WITH OPTIMIZED FUNCTIONALCRISPR-CAS SYSTEMS; U.S. application 62/054,651, 24 Sep. 14, DELIVERY,USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS ANDCOMPOSITIONS FOR MODELING COMPETITION OF MULTIPLE CANCER MUTATIONS INVIVO; U.S. application 62/067,886, 23 Oct. 14, DELIVERY, USE ANDTHERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FORMODELING COMPETITION OF MULTIPLE CANCER MUTATIONS IN VIVO; U.S.application 62/054,675, 24 Sep. 14, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS IN NEURONALCELLS/TISSUES; U.S. application 62/054,528, 24 Sep. 14, DELIVERY, USEAND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONSIN IMMUNE DISEASES OR DISORDERS; U.S. application 62/055,454, 25 Sep.14, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMSAND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING CELLPENETRATION PEPTIDES (CPP); U.S. application 62/055,460, 25 Sep. 14,MULTIFUNCTIONAL-CRISPR COMPLEXES AND/OR OPTIMIZED ENZYME LINKEDFUNCTIONAL-CRISPR COMPLEXES; U.S. application 62/087,475, 4 Dec. 14,FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S.application 62/055,487, 25 Sep. 14, FUNCTIONAL SCREENING WITH OPTIMIZEDFUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/087,546, 4 Dec. 14,MULTIFUNCTIONAL CRISPR COMPLEXES AND/OR OPTIMIZED ENZYME LINKEDFUNCTIONAL-CRISPR COMPLEXES; and U.S. application 62/098,285, 30 Dec.14, CRISPR MEDIATED IN VIVO MODELING AND GENETIC SCREENING OF TUMORGROWTH AND METASTASIS.

Each of these patents, patent publications, and applications, and alldocuments cited therein or during their prosecution (“appln citeddocuments”) and all documents cited or referenced in the appln citeddocuments, together with any instructions, descriptions, productspecifications, and product sheets for any products mentioned therein orin any document therein and incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. All documents (e.g., these patents, patent publicationsand applications and the appln cited documents) are incorporated hereinby reference to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by reference.

Also with respect to general information on CRISPR-Cas Systems, mentionis made of the following (also hereby incorporated herein by reference):

-   -   Multiplex genome engineering using CRISPR/Cas systems. Cong, L.,        Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.        D., Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. Science        February 15; 339(6121):819-23 (2013);    -   RNA guided editing of bacterial genomes using CRISPR-Cas        systems. Jiang W., Bikard D., Cox D., Zhang F, Marraffini L A.        Nat Biotechnol March; 31(3):233-9 (2013);    -   One-Step Generation of Mice Carrying Mutations in Multiple Genes        by CRISPR/Cas-Mediated Genome Engineering. Wang H., Yang H.,        Shivalila C S., Dawlaty M M., Cheng A W., Zhang F., Jaenisch R.        Cell May 9; 153(4):910-8 (2013);    -   Optical control of mammalian endogenous transcription and        epigenetic states. Konermann S, Brigham M D, Trevino A E, Hsu P        D, Heidenreich M, Cong L, Platt R J, Scott D A, Church G M,        Zhang F. Nature. 2013 Aug. 22; 500(7463):472-6. doi:        10.1038Nature12466. Epub 2013 Aug. 23;    -   Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome        Editing Specificity. Ran, F A., Hsu, P D., Lin, C Y.,        Gootenberg, J S., Konermann, S., Trevino, A E., Scott, D A.,        Inoue, A., Matoba, S., Zhang, Y., & Zhang, F. Cell August 28.        pii: S0092-8674(13)01015-5. (2013);    -   DNA targeting specificity of RNA-guided Cas9 nucleases. Hsu, P.,        Scott, D., Weinstein, J., Ran, F A., Konermann, S., Agarwala,        V., Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, T J.,        Marraffini, L A., Bao, G., & Zhang, F. Nat Biotechnol        doi:10.1038/nbt.2647 (2013);    -   Genome engineering using the CRISPR-Cas9 system. Ran, F A., Hsu,        P D., Wright, J., Agarwala, V., Scott, D A., Zhang, F. Nature        Protocols November; 8(11):2281-308. (2013);    -   Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells.        Shalem, O., Sanjana, N E., Hartenian, E., Shi, X., Scott, D A.,        Mikkelson, T., Heckl, D., Ebert, B L., Root, D E., Doench, J G.,        Zhang, F. Science December 12. (2013). [Epub ahead of print];    -   Crystal structure of cas9 in complex with guide RNA and target        DNA. Nishimasu, H., Ran, F A., Hsu, P D., Konermann, S.,        Shehata, S I., Dohmae, N., Ishitani, R., Zhang, F., Nureki, O.        Cell February 27. (2014). 156(5):935-49;    -   Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian        cells. Wu X., Scott D A., Kriz A J., Chiu A C., Hsu P D., Dadon        D B., Cheng A W., Trevino A E., Konermann S., Chen S., Jaenisch        R., Zhang F., Sharp P A. Nat Biotechnol. (2014) April 20. doi:        10.1038/nbt.2889,    -   CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling,        Platt et al., Cell 159(2): 440-455 (2014) DOI:        10.1016/j.cell.2014.09.014,    -   Development and Applications of CRISPR-Cas9 for Genome        Engineering, Hsu et al, Cell 157, 1262-1278 (Jun. 5, 2014) (Hsu        2014),    -   Genetic screens in human cells using the CRISPR/Cas9 system,        Wang et al., Science. 2014 Jan. 3; 343(6166): 80-84.        doi:10.1126/science.1246981,    -   Rational design of highly active sgRNAs for CRISPR-Cas9-mediated        gene inactivation, Doench et al., Nature Biotechnology published        online 3 Sep. 2014; doi:10.1038/nbt.3026, and    -   In vivo interrogation of gene function in the mammalian brain        using CRISPR-Cas9, Swiech et al, Nature Biotechnology; published        online 19 Oct. 2014; doi:10.1038/nbt.3055.    -   Genome-scale transcriptional activation by an engineered        CRISPR-Cas9 complex, Konermann S, Brigham M D, Trevino A E,        Joung J, Abudayyeh O O, Barcena C, Hsu P D, Habib N, Gootenberg        J S, Nishimasu H, Nureki O, Zhang F., Nature. January 29;        517(7536):583-8 (2015).    -   A split-Cas9 architecture for inducible genome editing and        transcription modulation, Zetsche B, Volz S E, Zhang F.,        (published online 2 Feb. 2015) Nat Biotechnol. February;        33(2):139-42 (2015);    -   Genome-wide CRISPR Screen in a Mouse Model of Tumor Growth and        Metastasis, Chen S, Sanjana N E, Zheng K, Shalem O, Lee K, Shi        X, Scott D A, Song J, Pan J Q, Weissleder R, Lee H, Zhang F,        Sharp P A. Cell 160, 1246-1260, Mar. 12, 2015 (multiplex screen        in mouse), and    -   In vivo genome editing using Staphylococcus aureus Cas9, Ran F        A, Cong L, Yan W X, Scott D A, Gootenberg J S, Kriz A J, Zetsche        B, Shalem O, Wu X, Makarova K S, Koonin E V, Sharp P A, Zhang        F., (published online 1 Apr. 2015), Nature. April 9;        520(7546):186-91 (2015).    -   High-throughput functional genomics using CRISPR-Cas9, Shalem et        al., Nature Reviews Genetics 16, 299-311 (May 2015).    -   Sequence determinants of improved CRISPR sgRNA design, Xu et        al., Genome Research 25, 1147-1157 (August 2015).    -   A Genome-wide CRISPR Screen in Primary Immune Cells to Dissect        Regulatory Networks, Parnas et al., Cell 162, 675-686 (Jul. 30,        2015).    -   CRISPR/Cas9 cleavage of viral DNA efficiently suppresses        hepatitis B virus, Ramanan et al., Scientific Reports 5:10833.        doi: 10.1038/srep10833 (Jun. 2, 2015).    -   Crystal Structure of Staphylococcus aureus Cas9, Nishimasu et        al., Cell 162, 1113-1126 (Aug. 27, 2015).    -   BCL11A enhancer dissection by Cas9-mediated in situ saturating        mutagenesis, Canver et al., Nature 527(7577):192-7 (Nov.        12, 2015) doi: 10.1038/nature15521. Epub 2015 Sep. 16. each of        which is incorporated herein by reference, and discussed briefly        below:    -   Cong et al. engineered type II CRISPR/Cas systems for use in        eukaryotic cells based on both Streptococcus thermophilus Cas9        and also Streptoccocus pyogenes Cas9 and demonstrated that Cas9        nucleases can be directed by short RNAs to induce precise        cleavage of DNA in human and mouse cells. Their study further        showed that Cas9 as converted into a nicking enzyme can be used        to facilitate homology-directed repair in eukaryotic cells with        minimal mutagenic activity. Additionally, their study        demonstrated that multiple guide sequences can be encoded into a        single CRISPR array to enable simultaneous editing of several at        endogenous genomic loci sites within the mammalian genome,        demonstrating easy programmability and wide applicability of the        RNA-guided nuclease technology. This ability to use RNA to        program sequence specific DNA cleavage in cells defined a new        class of genome engineering tools. These studies further showed        that other CRISPR loci are likely to be transplantable into        mammalian cells and can also mediate mammalian genome cleavage.        Importantly, it can be envisaged that several aspects of the        CRISPR/Cas system can be further improved to increase its        efficiency and versatility.    -   Jiang et al. used the clustered, regularly interspaced, short        palindromic repeats (CRISPR)—associated Cas9 endonuclease        complexed with dual-RNAs to introduce precise mutations in the        genomes of Streptococcus pneumoniae and Escherichia coli. The        approach relied on dual-RNA:Cas9-directed cleavage at the        targeted genomic site to kill unmutated cells and circumvents        the need for selectable markers or counter-selection systems.        The study reported reprogramming dual-RNA:Cas9 specificity by        changing the sequence of short CRISPR RNA (crRNA) to make        single- and multinucleotide changes carried on editing        templates. The study showed that simultaneous use of two crRNAs        enabled multiplex mutagenesis. Furthermore, when the approach        was used in combination with recombineering, in S. pneumoniae,        nearly 100% of cells that were recovered using the described        approach contained the desired mutation, and in E. coli, 65%        that were recovered contained the mutation.    -   Wang et al. (2013) used the CRISPR/Cas system for the one-step        generation of mice carrying mutations in multiple genes which        were traditionally generated in multiple steps by sequential        recombination in embryonic stem cells and/or time-consuming        intercrossing of mice with a single mutation. The CRISPR/Cas        system will greatly accelerate the in vivo study of functionally        redundant genes and of epistatic gene interactions.    -   Konermann et al. addressed the need in the art for versatile and        robust technologies that enable optical and chemical modulation        of DNA-binding domains based CRISPR Cas9 enzyme and also        Transcriptional Activator Like Effectors.    -   Ran et al. (2013-A) described an approach that combined a Cas9        nickase mutant with paired guide RNAs to introduce targeted        double-strand breaks. This addresses the issue of the Cas9        nuclease from the microbial CRISPR-Cas system being targeted to        specific genomic loci by a guide sequence, which can tolerate        certain mismatches to the DNA target and thereby promote        undesired off-target mutagenesis. Because individual nicks in        the genome are repaired with high fidelity, simultaneous nicking        via appropriately offset guide RNAs is required for        double-stranded breaks and extends the number of specifically        recognized bases for target cleavage. The authors demonstrated        that using paired nicking can reduce off-target activity by 50-        to 1,500-fold in cell lines and to facilitate gene knockout in        mouse zygotes without sacrificing on-target cleavage efficiency.        This versatile strategy enables a wide variety of genome editing        applications that require high specificity.    -   Hsu et al. (2013) characterized SpCas9 targeting specificity in        human cells to inform the selection of target sites and avoid        off-target effects. The study evaluated >700 guide RNA variants        and SpCas9-induced indel mutation levels at >100 predicted        genomic off-target loci in 293T and 293FT cells. The authors        that SpCas9 tolerates mismatches between guide RNA and target        DNA at different positions in a sequence-dependent manner,        sensitive to the number, position and distribution of        mismatches. The authors further showed that SpCas9-mediated        cleavage is unaffected by DNA methylation and that the dosage of        SpCas9 and sgRNA can be titrated to minimize off-target        modification. Additionally, to facilitate mammalian genome        engineering applications, the authors reported providing a        web-based software tool to guide the selection and validation of        target sequences as well as off-target analyses.    -   Ran et al. (2013-B) described a set of tools for Cas9-mediated        genome editing via non-homologous end joining (NHEJ) or        homology-directed repair (HDR) in mammalian cells, as well as        generation of modified cell lines for downstream functional        studies. To minimize off-target cleavage, the authors further        described a double-nicking strategy using the Cas9 nickase        mutant with paired guide RNAs. The protocol provided by the        authors experimentally derived guidelines for the selection of        target sites, evaluation of cleavage efficiency and analysis of        off-target activity. The studies showed that beginning with        target design, gene modifications can be achieved within as        little as 1-2 weeks, and modified clonal cell lines can be        derived within 2-3 weeks.    -   Shalem et al. described a new way to interrogate gene function        on a genome-wide scale. Their studies showed that delivery of a        genome-scale CRISPR-Cas9 knockout (GeCKO) library targeted        18,080 genes with 64,751 unique guide sequences enabled both        negative and positive selection screening in human cells. First,        the authors showed use of the GeCKO library to identify genes        essential for cell viability in cancer and pluripotent stem        cells. Next, in a melanoma model, the authors screened for genes        whose loss is involved in resistance to vemurafenib, a        therapeutic that inhibits mutant protein kinase BRAF. Their        studies showed that the highest-ranking candidates included        previously validated genes NF1 and MED12 as well as novel hits        NF2, CUL3, TADA2B, and TADA1. The authors observed a high level        of consistency between independent guide RNAs targeting the same        gene and a high rate of hit confirmation, and thus demonstrated        the promise of genome-scale screening with Cas9.    -   Nishimasu et al. reported the crystal structure of Streptococcus        pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A°        resolution. The structure revealed a bilobed architecture        composed of target recognition and nuclease lobes, accommodating        the sgRNA:DNA heteroduplex in a positively charged groove at        their interface. Whereas the recognition lobe is essential for        binding sgRNA and DNA, the nuclease lobe contains the HNH and        RuvC nuclease domains, which are properly positioned for        cleavage of the complementary and non-complementary strands of        the target DNA, respectively. The nuclease lobe also contains a        carboxyl-terminal domain responsible for the interaction with        the protospacer adjacent motif (PAM). This high-resolution        structure and accompanying functional analyses have revealed the        molecular mechanism of RNA-guided DNA targeting by Cas9, thus        paving the way for the rational design of new, versatile        genome-editing technologies.    -   Wu et al. mapped genome-wide binding sites of a catalytically        inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with        single guide RNAs (sgRNAs) in mouse embryonic stem cells        (mESCs). The authors showed that each of the four sgRNAs tested        targets dCas9 to between tens and thousands of genomic sites,        frequently characterized by a 5-nucleotide seed region in the        sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin        inaccessibility decreases dCas9 binding to other sites with        matching seed sequences; thus 70% of off-target sites are        associated with genes. The authors showed that targeted        sequencing of 295 dCas9 binding sites in mESCs transfected with        catalytically active Cas9 identified only one site mutated above        background levels. The authors proposed a two-state model for        Cas9 binding and cleavage, in which a seed match triggers        binding but extensive pairing with target DNA is required for        cleavage.    -   Platt et al. established a Cre-dependent Cas9 knockin mouse. The        authors demonstrated in vivo as well as ex vivo genome editing        using adeno-associated virus (AAV)-, lentivirus-, or        particle-mediated delivery of guide RNA in neurons, immune        cells, and endothelial cells.    -   Hsu et al. (2014) is a review article that discusses generally        CRISPR-Cas9 history from yogurt to genome editing, including        genetic screening of cells.    -   Wang et al. (2014) relates to a pooled, loss-of-function genetic        screening approach suitable for both positive and negative        selection that uses a genome-scale lentiviral single guide RNA        (sgRNA) library.    -   Doench et al. created a pool of sgRNAs, tiling across all        possible target sites of a panel of six endogenous mouse and        three endogenous human genes and quantitatively assessed their        ability to produce null alleles of their target gene by antibody        staining and flow cytometry. The authors showed that        optimization of the PAM improved activity and also provided an        on-line tool for designing sgRNAs.    -   Swiech et al. demonstrate that AAV-mediated SpCas9 genome        editing can enable reverse genetic studies of gene function in        the brain.    -   Konermann et al. (2015) discusses the ability to attach multiple        effector domains, e.g., transcriptional activator, functional        and epigenomic regulators at appropriate positions on the guide        such as stem or tetraloop with and without linkers.    -   Zetsche et al. demonstrates that the Cas9 enzyme can be split        into two and hence the assembly of Cas9 for activation can be        controlled.    -   Chen et al. relates to multiplex screening by demonstrating that        a genome-wide in vivo CRISPR-Cas9 screen in mice reveals genes        regulating lung metastasis.    -   Ran et al. (2015) relates to SaCas9 and its ability to edit        genomes and demonstrates that one cannot extrapolate from        biochemical assays. Shalem et al. (2015) described ways in which        catalytically inactive Cas9 (dCas9) fusions are used to        synthetically repress (CRISPRi) or activate (CRISPRa)        expression, showing. advances using Cas9 for genome-scale        screens, including arrayed and pooled screens, knockout        approaches that inactivate genomic loci and strategies that        modulate transcriptional activity.    -   Shalem et al. (2015) described ways in which catalytically        inactive Cas9 (dCas9) fusions are used to synthetically repress        (CRISPRi) or activate (CRISPRa) expression, showing. advances        using Cas9 for genome-scale screens, including arrayed and        pooled screens, knockout approaches that inactivate genomic loci        and strategies that modulate transcriptional activity.    -   Xu et al. (2015) assessed the DNA sequence features that        contribute to single guide RNA (sgRNA) efficiency in        CRISPR-based screens. The authors explored efficiency of        CRISPR/Cas9 knockout and nucleotide preference at the cleavage        site. The authors also found that the sequence preference for        CRISPRi/a is substantially different from that for CRISPR/Cas9        knockout.    -   Parnas et al. (2015) introduced genome-wide pooled CRISPR-Cas9        libraries into dendritic cells (DCs) to identify genes that        control the induction of tumor necrosis factor (Tnf) by        bacterial lipopolysaccharide (LPS). Known regulators of Tlr4        signaling and previously unknown candidates were identified and        classified into three functional modules with distinct effects        on the canonical responses to LPS.    -   Ramanan et al (2015) demonstrated cleavage of viral episomal DNA        (cccDNA) in infected cells. The HBV genome exists in the nuclei        of infected hepatocytes as a 3.2 kb double-stranded episomal DNA        species called covalently closed circular DNA (cccDNA), which is        a key component in the HBV life cycle whose replication is not        inhibited by current therapies. The authors showed that sgRNAs        specifically targeting highly conserved regions of HBV robustly        suppresses viral replication and depleted cccDNA.    -   Nishimasu et al. (2015) reported the crystal structures of        SaCas9 in complex with a single guide RNA (sgRNA) and its        double-stranded DNA targets, containing the 5′-TTGAAT-3′ PAM and        the 5′-TTGGGT-3′ PAM. A structural comparison of SaCas9 with        SpCas9 highlighted both structural conservation and divergence,        explaining their distinct PAM specificities and orthologous        sgRNA recognition.    -   Slaymaker et al (2015) reported the use of structure-guided        protein engineering to improve the specificity of Streptococcus        pyogenes Cas9 (SpCas9). The authors developed “enhanced        specificity” SpCas9 (eSpCas9) variants which maintained robust        on-target cleavage with reduced off-target effects.

Mention is also made of Tsai et al, “Dimeric CRISPR RNA-guided FokInucleases for highly specific genome editing,” Nature Biotechnology32(6): 569-77 (2014) which is not believed to be prior art to theinstant invention or application, but which may be considered in thepractice of the instant invention. Mention is also made of Konermann etal., “Genome-scale transcription activation by an engineered CRISPR-Cas9complex,” doi:10.1038/nature14136, incorporated herein by reference.

In general, the CRISPR-Cas or CRISPR system is as used in the foregoingdocuments, such as WO 2014/093622 (PCT/US2013/074667) and referscollectively to transcripts and other elements involved in theexpression of or directing the activity of CRISPR-associated (“Cas”)genes, including sequences encoding a Cas gene, a tracr(trans-activating CRISPR) sequence (e.g. tracrRNA or an active partialtracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and atracrRNA-processed partial direct repeat in the context of an endogenousCRISPR system), a guide sequence (also referred to as a “spacer” in thecontext of an endogenous CRISPR system), or “RNA(s)” as that term isherein used (e.g., RNA(s) to guide Cas9, e.g. CRISPR RNA andtransactivating (tracr) RNA or a single guide RNA (sgRNA) (chimericRNA)) or other sequences and transcripts from a CRISPR locus. Ingeneral, a CRISPR system is characterized by elements that promote theformation of a CRISPR complex at the site of a target sequence (alsoreferred to as a protospacer in the context of an endogenous CRISPRsystem). In the context of formation of a CRISPR complex, “targetsequence” refers to a sequence to which a guide sequence is designed tohave complementarity, where hybridization between a target sequence anda guide sequence promotes the formation of a CRISPR complex. A targetsequence may comprise any polynucleotide, such as DNA or RNApolynucleotides. In some embodiments, a target sequence is located inthe nucleus or cytoplasm of a cell. In some embodiments, direct repeatsmay be identified in silico by searching for repetitive motifs thatfulfill any or all of the following criteria: 1. found in a 2 Kb windowof genomic sequence flanking the type II CRISPR locus; 2. span from 20to 50 bp; and 3. interspaced by 20 to 50 bp. In some embodiments, 2 ofthese criteria may be used, for instance 1 and 2, 2 and 3, or 1 and 3.In some embodiments, all 3 criteria may be used. In some embodiments itmay be preferred in a CRISPR complex that the tracr sequence has one ormore hairpins and is 30 or more nucleotides in length, 40 or morenucleotides in length, or 50 or more nucleotides in length; the guidesequence is between 10 to 30 nucleotides in length, the CRISPR/Casenzyme is a Type II Cas9 enzyme. In embodiments of the invention theterms guide sequence and guide RNA are used interchangeably as inforegoing cited documents such as WO 2014/093622 (PCT/US2013/074667). Ingeneral, a guide sequence is any polynucleotide sequence havingsufficient complementarity with a target polynucleotide sequence tohybridize with the target sequence and direct sequence-specific bindingof a CRISPR complex to the target sequence. In some embodiments, thedegree of complementarity between a guide sequence and its correspondingtarget sequence, when optimally aligned using a suitable alignmentalgorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%,95%, 97.5%, 99%, or more. Optimal alignment may be determined with theuse of any suitable algorithm for aligning sequences, non-limitingexample of which include the Smith-Waterman algorithm, theNeedleman-Wunsch algorithm, algorithms based on the Burrows-WheelerTransform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT,Novoalign (Novocraft Technologies; available at www.novocraft.com),ELAND (Illumina, San Diego, Calif.), SOAP (available atsoap.genomics.org.cn), and Maq (available at maq.sourceforge.net). Insome embodiments, a guide sequence is about or more than about 5, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In someembodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30,25, 20, 15, 12, or fewer nucleotides in length. Preferably the guidesequence is 10-30 nucleotides long. The ability of a guide sequence todirect sequence-specific binding of a CRISPR complex to a targetsequence may be assessed by any suitable assay. For example, thecomponents of a CRISPR system sufficient to form a CRISPR complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target sequence, such as by transfectionwith vectors encoding the components of the CRISPR sequence, followed byan assessment of preferential cleavage within the target sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget polynucleotide sequence may be evaluated in a test tube byproviding the target sequence, components of a CRISPR complex, includingthe guide sequence to be tested and a control guide sequence differentfrom the test guide sequence, and comparing binding or rate of cleavageat the target sequence between the test and control guide sequencereactions. Other assays are possible, and will occur to those skilled inthe art.A guide sequence may be selected to target any target sequence.In some embodiments, the target sequence is a sequence within a genomeof a cell. Exemplary target sequences include those that are unique inthe target genome. For example, for the S. pyogenes Cas9, a uniquetarget sequence in a genome may include a Cas9 target site of the formMMMMMMMMNNNNNNNNNNNNXGG (SEQ ID NO: 1) where NNNNNNNNNNNNXGG (SEQ ID NO:2) (N is A, G, T, or C; and X can be anything) has a single occurrencein the genome. A unique target sequence in a genome may include an S.pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNXGG (SEQ ID NO:3) where NNNNNNNNNNNXGG (SEQ ID NO: 4) (N is A, G, T, or C; and X can beanything) has a single occurrence in the genome. For the S. thermophilusCRISPR1 Cas9, a unique target sequence in a genome may include a Cas9target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW (SEQ ID NO: 5) whereNNNNNNNNNNNNXXAGAAW (SEQ ID NO: 6) (N is A, G, T, or C; X can beanything; and W is A or T) has a single occurrence in the genome. Aunique target sequence in a genome may include an S. thermophilusCRISPR1 Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXXAGAAW (SEQ IDNO: 7) where NNNNNNNNNNNXXAGAAW (SEQ ID NO: 8) (N is A, G, T, or C; Xcan be anything; and W is A or T) has a single occurrence in the genome.For the S. pyogenes Cas9, a unique target sequence in a genome mayinclude a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGGXG (SEQ IDNO: 9) where NNNNNNNNNNNNXGGXG (SEQ ID NO: 10) (N is A, G, T, or C; andX can be anything) has a single occurrence in the genome. A uniquetarget sequence in a genome may include an S. pyogenes Cas9 target siteof the form MMMMMMMMMNNNNNNNNNNNXGGXG (SEQ ID NO: 11) whereNNNNNNNNNNNXGGXG (SEQ ID NO: 12) (N is A, G, T, or C; and X can beanything) has a single occurrence in the genome. In each of thesesequences “M” may be A, G, T, or C, and need not be considered inidentifying a sequence as unique. In some embodiments, a guide sequenceis selected to reduce the degree secondary structure within the guidesequence. In some embodiments, about or less than about 75%, 50%, 40%,30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of theguide sequence participate in self-complementary base pairing whenoptimally folded. Optimal folding may be determined by any suitablepolynucleotide folding algorithm. Some programs are based on calculatingthe minimal Gibbs free energy. An example of one such algorithm ismFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981),133-148). Another example folding algorithm is the online webserverRNAfold, developed at Institute for Theoretical Chemistry at theUniversity of Vienna, using the centroid structure prediction algorithm(see e.g. A. R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr andGM Church, 2009, Nature Biotechnology 27(12): 1151-62).

In general, a tracr mate sequence includes any sequence that hassufficient complementarity with a tracr sequence to promote one or moreof: (1) excision of a guide sequence flanked by tracr mate sequences ina cell containing the corresponding tracr sequence; and (2) formation ofa CRISPR complex at a target sequence, wherein the CRISPR complexcomprises the tracr mate sequence hybridized to the tracr sequence. Ingeneral, degree of complementarity is with reference to the optimalalignment of the tracr mate sequence and tracr sequence, along thelength of the shorter of the two sequences. Optimal alignment may bedetermined by any suitable alignment algorithm, and may further accountfor secondary structures, such as self-complementarity within either thetracr sequence or tracr mate sequence. In some embodiments, the degreeof complementarity between the tracr sequence and tracr mate sequencealong the length of the shorter of the two when optimally aligned isabout or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,97.5%, 99%, or higher. In some embodiments, the tracr sequence is aboutor more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 40, 50, or more nucleotides in length. In someembodiments, the tracr sequence and tracr mate sequence are containedwithin a single transcript, such that hybridization between the twoproduces a transcript having a secondary structure, such as a hairpin.In an embodiment of the invention, the transcript or transcribedpolynucleotide sequence has at least two or more hairpins. In preferredembodiments, the transcript has two, three, four or five hairpins. In afurther embodiment of the invention, the transcript has at most fivehairpins. In a hairpin structure the portion of the sequence 5′ of thefinal “N” and upstream of the loop corresponds to the tracr matesequence, and the portion of the sequence 3′ of the loop corresponds tothe tracr sequence Further non-limiting examples of singlepolynucleotides comprising a guide sequence, a tracr mate sequence, anda tracr sequence are as follows (listed 5′ to 3′), where “N” representsa base of a guide sequence, the first block of lower case lettersrepresent the tracr mate sequence, and the second block of lower caseletters represent the tracr sequence, and the final poly-T sequencerepresents the transcription terminator: (1)NNNNNNNNNNNNNNNNNNNNgrrrrrgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ IDNO: 13); (2)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 14);(3)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgtTTTTTT (SEQ ID NO: 15); (4)NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTT (SEQ ID NO: 16); (5)NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaacttgaaaaagtgTTTTTTT (SEQ ID NO: 17); and (6)NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTTTTTTT (SEQ ID NO: 18). In some embodiments, sequences (1) to (3) areused in combination with Cas9 from S. thermophilus CRISPR1. In someembodiments, sequences (4) to (6) are used in combination with Cas9 fromS. pyogenes. In some embodiments, the tracr sequence is a separatetranscript from a transcript comprising the tracr mate sequence.

In some embodiments, candidate tracrRNA may be subsequently predicted bysequences that fulfill any or all of the following criteria: 1. sequencehomology to direct repeats (motif search in Geneious with up to 18-bpmismatches); 2. presence of a predicted Rho-independent transcriptionalterminator in direction of transcription; and 3. stable hairpinsecondary structure between tracrRNA and direct repeat. In someembodiments, 2 of these criteria may be used, for instance 1 and 2, 2and 3, or 1 and 3. In some embodiments, all 3 criteria may be used.

In some embodiments, chimeric synthetic guide RNAs (sgRNAs) designs mayincorporate at least 12 bp of duplex structure between the direct repeatand tracrRNA.

For minimization of toxicity and off-target effect, it will be importantto control the concentration of CRISPR enzyme mRNA and guide RNAdelivered. Optimal concentrations of CRISPR enzyme mRNA and guide RNAcan be determined by testing different concentrations in a cellular ornon-human eukaryote animal model and using deep sequencing the analyzethe extent of modification at potential off-target genomic loci. Forexample, for the guide sequence targeting 5′-GAGTCCGAGCAGAAGAAGAA-3′(SEQ ID NO: 19) in the EMX1 gene of the human genome, deep sequencingcan be used to assess the level of modification at the following twooff-target loci, 1: 5′-GAGTCCTAGCAGGAGAAGAA-3′ (SEQ ID NO: 20) and 2:5′-GAGTCTAAGCAGAAGAAGAA-3′ (SEQ ID NO: 21). The concentration that givesthe highest level of on-target modification while minimizing the levelof off-target modification should be chosen for in vivo delivery.Alternatively, to minimize the level of toxicity and off-target effect,CRISPR enzyme nickase mRNA (for example S. pyogenes Cas9 with the D10Amutation) can be delivered with a pair of guide RNAs targeting a site ofinterest. The two guide RNAs need to be spaced as follows. Guidesequences and strategies to mimize toxicity and off-target effects canbe as in WO 2014/093622 (PCT/US2013/074667).

The CRISPR system is derived advantageously from a type II CRISPRsystem. In some embodiments, one or more elements of a CRISPR system isderived from a particular organism comprising an endogenous CRISPRsystem, such as Streptococcus pyogenes. In preferred embodiments of theinvention, the CRISPR system is a type II CRISPR system and the Casenzyme is Cas9, which catalyzes DNA cleavage. Non-limiting examples ofCas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7,Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3,Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1,Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16,CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologues thereof, ormodified versions thereof.

In some embodiments, the unmodified CRISPR enzyme has DNA cleavageactivity, such as Cas9. In some embodiments, the CRISPR enzyme directscleavage of one or both strands at the location of a target sequence,such as within the target sequence and/or within the complement of thetarget sequence. In some embodiments, the CRISPR enzyme directs cleavageof one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 50, 100, 200, 500, or more base pairs from the first or lastnucleotide of a target sequence. In some embodiments, a vector encodes aCRISPR enzyme that is mutated to with respect to a correspondingwild-type enzyme such that the mutated CRISPR enzyme lacks the abilityto cleave one or both strands of a target polynucleotide containing atarget sequence. For example, an aspartate-to-alanine substitution(D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes convertsCas9 from a nuclease that cleaves both strands to a nickase (cleaves asingle strand). Other examples of mutations that render Cas9 a nickaseinclude, without limitation, H840A, N854A, and N863A. As a furtherexample, two or more catalytic domains of Cas9 (RuvC I, RuvC II, andRuvC III or the HNH domain) may be mutated to produce a mutated Cas9substantially lacking all DNA cleavage activity. In some embodiments, aD10A mutation is combined with one or more of H840A, N854A, or N863Amutations to produce a Cas9 enzyme substantially lacking all DNAcleavage activity. In some embodiments, a CRISPR enzyme is considered tosubstantially lack all DNA cleavage activity when the DNA cleavageactivity of the mutated enzyme is about no more than 25%, 10%, 5%, 1%,0.1%, 0.01%, or less of the DNA cleavage activity of the non-mutatedform of the enzyme; an example can be when the DNA cleavage activity ofthe mutated form is nil or negligible as compared with the non-mutatedform. Where the enzyme is not SpCas9, mutations may be made at any orall residues corresponding to positions 10, 762, 840, 854, 863 and/or986 of SpCas9 (which may be ascertained for instance by standardsequence comparison tools). In particular, any or all of the followingmutations are preferred in SpCas9: D10A, E762A, H840A, N854A, N863Aand/or D986A; as well as conservative substitution for any of thereplacement amino acids is also envisaged. The same (or conservativesubstitutions of these mutations) at corresponding positions in otherCas9s are also preferred. Particularly preferred are D10 and H840 inSpCas9. However, in other Cas9s, residues corresponding to SpCas9 D10and H840 are also preferred. For instance, an N580 or N580A mutation inSaCas9. Orthologs of SpCas9 can be used in the practice of theinvention. A Cas enzyme may be identified Cas9 as this can refer to thegeneral class of enzymes that share homology to the biggest nucleasewith multiple nuclease domains from the type II CRISPR system. Mostpreferably, the Cas9 enzyme is from, or is derived from, spCas9 (S.pyogenes Cas9) or saCas9 (S. aureus Cas9). StCas9″ refers to wild typeCas9 from S. thermophilus, the protein sequence of which is given in theSwissProt database under accession number G3ECR1. Similarly, S pyogenesCas9 or spCas9 is included in SwissProt under accession number Q99ZW2.By derived, Applicants mean that the derived enzyme is largely based, inthe sense of having a high degree of sequence homology with, a wildtypeenzyme, but that it has been mutated (modified) in some way as describedherein. It will be appreciated that the terms Cas and CRISPR enzyme aregenerally used herein interchangeably, unless otherwise apparent. Asmentioned above, many of the residue numberings used herein refer to theCas9 enzyme from the type II CRISPR locus in Streptococcus pyogenes.However, it will be appreciated that this invention includes many moreCas9s from other species of microbes, such as SpCas9, SaCa9, St1Cas9 andso forth. Enzymatic action by Cas9 derived from Streptococcus pyogenesor any closely related Cas9 generates double stranded breaks at targetsite sequences which hybridize to 20 nucleotides of the guide sequenceand that have a protospacer-adjacent motif (PAM) sequence (examplesinclude NGG/NRG or a PAM that can be determined as described herein)following the 20 nucleotides of the target sequence. CRISPR activitythrough Cas9 for site-specific DNA recognition and cleavage is definedby the guide sequence, the tracr sequence that hybridizes in part to theguide sequence and the PAM sequence. More aspects of the CRISPR systemare described in Karginov and Hannon, The CRISPR system: smallRNA-guided defence in bacteria and archaea, Mole Cell 2010, Jan. 15;37(1): 7. The type II CRISPR locus from Streptococcus pyogenes SF370,which contains a cluster of four genes Cas9, Cas1, Cas2, and Csn1, aswell as two non-coding RNA elements, tracrRNA and a characteristic arrayof repetitive sequences (direct repeats) interspaced by short stretchesof non-repetitive sequences (spacers, about 30 bp each). In this system,targeted DNA double-strand break (DSB) is generated in four sequentialsteps. First, two non-coding RNAs, the pre-crRNA array and tracrRNA, aretranscribed from the CRISPR locus. Second, tracrRNA hybridizes to thedirect repeats of pre-crRNA, which is then processed into mature crRNAscontaining individual spacer sequences. Third, the mature crRNA:tracrRNAcomplex directs Cas9 to the DNA target consisting of the protospacer andthe corresponding PAM via heteroduplex formation between the spacerregion of the crRNA and the protospacer DNA. Finally, Cas9 mediatescleavage of target DNA upstream of PAM to create a DSB within theprotospacer. A pre-crRNA array consisting of a single spacer flanked bytwo direct repeats (DRs) is also encompassed by the term “tracr-matesequences”). In certain embodiments, Cas9 may be constitutively presentor inducibly present or conditionally present or administered ordelivered. Cas9 optimization may be used to enhance function or todevelop new functions, one can generate chimeric Cas9 proteins. And Cas9may be used as a generic DNA binding protein.

Typically, in the context of an endogenous CRISPR system, formation of aCRISPR complex (comprising a guide sequence hybridized to a targetsequence and complexed with one or more Cas proteins) results incleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence.Without wishing to be bound by theory, the tracr sequence, which maycomprise or consist of all or a portion of a wild-type tracr sequence(e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, ormore nucleotides of a wild-type tracr sequence), may also form part of aCRISPR complex, such as by hybridization along at least a portion of thetracr sequence to all or a portion of a tracr mate sequence that isoperably linked to the guide sequence.

An example of a codon optimized sequence, is in this instance a sequenceoptimized for expression in a eukaryote, e.g., humans (i.e. beingoptimized for expression in humans), or for another eukaryote, animal ormammal as herein discussed; see, e.g., SaCas9 human codon optimizedsequence in WO 2014/093622 (PCT/US2013/074667). Whilst this ispreferred, it will be appreciated that other examples are possible andcodon optimization for a host species other than human, or for codonoptimization for specific organs is known. In some embodiments, anenzyme coding sequence encoding a CRISPR enzyme is codon optimized forexpression in particular cells, such as eukaryotic cells. The eukaryoticcells may be those of or derived from a particular organism, such as amammal, including but not limited to human, or non-human eukaryote oranimal or mammal as herein discussed, e.g., mouse, rat, rabbit, dog,livestock, or non-human mammal or primate. In some embodiments,processes for modifying the germ line genetic identity of human beingsand/or processes for modifying the genetic identity of animals which arelikely to cause them suffering without any substantial medical benefitto man or animal, and also animals resulting from such processes, may beexcluded. In general, codon optimization refers to a process ofmodifying a nucleic acid sequence for enhanced expression in the hostcells of interest by replacing at least one codon (e.g. about or morethan about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of thenative sequence with codons that are more frequently or most frequentlyused in the genes of that host cell while maintaining the native aminoacid sequence. Various species exhibit particular bias for certaincodons of a particular amino acid. Codon bias (differences in codonusage between organisms) often correlates with the efficiency oftranslation of messenger RNA (mRNA), which is in turn believed to bedependent on, among other things, the properties of the codons beingtranslated and the availability of particular transfer RNA (tRNA)molecules. The predominance of selected tRNAs in a cell is generally areflection of the codons used most frequently in peptide synthesis.Accordingly, genes can be tailored for optimal gene expression in agiven organism based on codon optimization. Codon usage tables arereadily available, for example, at the “Codon Usage Database” availableat www.kazusa.orjp/codon/ and these tables can be adapted in a number ofways. See Nakamura, Y., et al. “Codon usage tabulated from theinternational DNA sequence databases: status for the year 2000” Nucl.Acids Res. 28:292 (2000). Computer algorithms for codon optimizing aparticular sequence for expression in a particular host cell are alsoavailable, such as Gene Forge (Aptagen; Jacobus, Pa.), are alsoavailable. In some embodiments, one or more codons (e.g. 1, 2, 3, 4, 5,10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding aCRISPR enzyme correspond to the most frequently used codon for aparticular amino acid.

In some embodiments, a vector encodes a CRISPR enzyme comprising one ormore nuclear localization sequences (NLSs), such as about or more thanabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments,the CRISPR enzyme comprises about or more than about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near thecarboxy-terminus, or a combination of these (e.g. zero or at least oneor more NLS at the amino-terminus and zero or at one or more NLS at thecarboxy terminus). When more than one NLS is present, each may beselected independently of the others, such that a single NLS may bepresent in more than one copy and/or in combination with one or moreother NLSs present in one or more copies. In a preferred embodiment ofthe invention, the CRISPR enzyme comprises at most 6 NLSs. In someembodiments, an NLS is considered near the N- or C-terminus when thenearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20,25, 30, 40, 50, or more amino acids along the polypeptide chain from theN- or C-terminus. Non-limiting examples of NLSs include an NLS sequencederived from: the NLS of the SV40 virus large T-antigen, having theamino acid sequence PKKKRKV (SEQ ID NO: 22); the NLS from nucleoplasmin(e.g. the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK(SEQ ID NO: 23)); the c-myc NLS having the amino acid sequence PAAKRVKLD(SEQ ID NO: 24) or RQRRNELKRSP (SEQ ID NO: 25); the hRNPA1 M9 NLS havingthe sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 26); thesequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 27) ofthe IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO:28) and PPKKARED (SEQ ID NO: 29) of the myoma T protein; the sequencePQPKKKPL (SEQ ID NO: 30) of human p53; the sequence SALIKKKKKMAP (SEQ IDNO: 31) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 32) andPKQKKRK (SEQ ID NO: 33) of the influenza virus NS1; the sequenceRKLKKKIKKL (SEQ ID NO: 34) of the Hepatitis virus delta antigen; thesequence REKKKFLKRR (SEQ ID NO: 35) of the mouse Mx1 protein; thesequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 36) of the humanpoly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ IDNO: 37) of the steroid hormone receptors (human) glucocorticoid. Ingeneral, the one or more NLSs are of sufficient strength to driveaccumulation of the CRISPR enzyme in a detectable amount in the nucleusof a eukaryotic cell. In general, strength of nuclear localizationactivity may derive from the number of NLSs in the CRISPR enzyme, theparticular NLS(s) used, or a combination of these factors. Detection ofaccumulation in the nucleus may be performed by any suitable technique.For example, a detectable marker may be fused to the CRISPR enzyme, suchthat location within a cell may be visualized, such as in combinationwith a means for detecting the location of the nucleus (e.g. a stainspecific for the nucleus such as DAPI). Cell nuclei may also be isolatedfrom cells, the contents of which may then be analyzed by any suitableprocess for detecting protein, such as immunohistochemistry, Westernblot, or enzyme activity assay. Accumulation in the nucleus may also bedetermined indirectly, such as by an assay for the effect of CRISPRcomplex formation (e.g. assay for DNA cleavage or mutation at the targetsequence, or assay for altered gene expression activity affected byCRISPR complex formation and/or CRISPR enzyme activity), as compared toa control no exposed to the CRISPR enzyme or complex, or exposed to aCRISPR enzyme lacking the one or more NLSs.

Aspects of the invention relate to the expression of the gene productbeing decreased or a template polynucleotide being further introducedinto a DNA or RNA molecule encoding the gene product or an interveningsequence being excised precisely by allowing the two 5′ overhangs toreanneal and ligate or the activity or function of the gene productbeing altered or the expression of the gene product being increased. Inan embodiment of the invention, the gene product is a protein. Incertain Cas9 systems, sgRNA pairs creating 5′ overhangs with less than 8bp overlap between the guide sequences (offset greater than −8 bp) wereable to mediate detectable indel formation; and each guide used in theseassays is able to efficiently induce indels when paired with wildtypeCas9, indicating that the relative positions of the guide pairs are themost important parameters in predicting double nicking activity. SinceCas9n and Cas9H840A nick opposite strands of DNA, substitution of Cas9nwith Cas9H840A with a given sgRNA pair should have resulted in theinversion of the overhang type; but no indel formation is observed aswith Cas9H840A indicating that Cas9H840A is a CRISPR enzymesubstantially lacking all DNA cleavage activity (which is when the DNAcleavage activity of the mutated enzyme is about no more than 25%, 10%,5%, 1%, 0.1%, 0.01%, or less of the DNA cleavage activity of thenon-mutated form of the enzyme; whereby an example can be when the DNAcleavage activity of the mutated form is nil or negligible as comparedwith the non-mutated form, e.g., when no indel formation is observed aswith Cas9H840A in the eukaryotic system in contrast to the biochemicalor prokaryotic systems). Nonetheless, a pair of sgRNAs that willgenerate a 5′ overhang with Cas9n should in principle generate thecorresponding 3′ overhang instead, and double nicking. Therefore, sgRNApairs that lead to the generation of a 3′ overhang with Cas9n can beused with another mutated Cas9 to generate a 5′ overhang, and doublenicking. Accordingly, in some embodiments, a recombination template isalso provided. A recombination template may be a component of anothervector as described herein, contained in a separate vector, or providedas a separate polynucleotide. In some embodiments, a recombinationtemplate is designed to serve as a template in homologous recombination,such as within or near a target sequence nicked or cleaved by a CRISPRenzyme as a part of a CRISPR complex. A template polynucleotide may beof any suitable length, such as about or more than about 10, 15, 20, 25,50, 75, 100, 150, 200, 500, 1000, or more nucleotides in length. In someembodiments, the template polynucleotide is complementary to a portionof a polynucleotide comprising the target sequence. When optimallyaligned, a template polynucleotide might overlap with one or morenucleotides of a target sequences (e.g. about or more than about 1, 5,10, 15, 20, or more nucleotides). In some embodiments, when a templatesequence and a polynucleotide comprising a target sequence are optimallyaligned, the nearest nucleotide of the template polynucleotide is withinabout 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000,10000, or more nucleotides from the target sequence.

In some embodiments, one or more vectors driving expression of one ormore elements of a CRISPR system are introduced into a host cell suchthat expression of the elements of the CRISPR system direct formation ofa CRISPR complex at one or more target sites. For example, a Cas enzyme,a guide sequence linked to a tracr-mate sequence, and a tracr sequencecould each be operably linked to separate regulatory elements onseparate vectors. Or, RNA(s) of the CRISPR System can be delivered to atransgenic Cas9 animal or mammal, e.g., an animal or mammal thatconstitutively or inducibly or conditionally expresses Cas9; or ananimal or mammal that is otherwise expressing Cas9 or has cellscontaining Cas9, such as by way of prior administration thereto of avector or vectors that code for and express in vivo Cas9. Alternatively,two or more of the elements expressed from the same or differentregulatory elements, may be combined in a single vector, with one ormore additional vectors providing any components of the CRISPR systemnot included in the first vector. CRISPR system elements that arecombined in a single vector may be arranged in any suitable orientation,such as one element located 5′ with respect to (“upstream” of) or 3′with respect to (“downstream” of) a second element. The coding sequenceof one element may be located on the same or opposite strand of thecoding sequence of a second element, and oriented in the same oropposite direction. In some embodiments, a single promoter drivesexpression of a transcript encoding a CRISPR enzyme and one or more ofthe guide sequence, tracr mate sequence (optionally operably linked tothe guide sequence), and a tracr sequence embedded within one or moreintron sequences (e.g. each in a different intron, two or more in atleast one intron, or all in a single intron). In some embodiments, theCRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequenceare operably linked to and expressed from the same promoter. Deliveryvehicles, vectors, particles, nanoparticles, formulations and componentsthereof for expression of one or more elements of a CRISPR system are asused in herein-cited documents, such as WO 2014/093622(PCT/US2013/074667). In some embodiments, a vector comprises one or moreinsertion sites, such as a restriction endonuclease recognition sequence(also referred to as a “cloning site”). In some embodiments, one or moreinsertion sites (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more insertion sites) are located upstream and/or downstreamof one or more sequence elements of one or more vectors. In someembodiments, a vector comprises an insertion site upstream of a tracrmate sequence, and optionally downstream of a regulatory elementoperably linked to the tracr mate sequence, such that followinginsertion of a guide sequence into the insertion site and uponexpression the guide sequence directs sequence-specific binding of aCRISPR complex to a target sequence in a eukaryotic cell. In someembodiments, a vector comprises two or more insertion sites, eachinsertion site being located between two tracr mate sequences so as toallow insertion of a guide sequence at each site. In such anarrangement, the two or more guide sequences may comprise two or morecopies of a single guide sequence, two or more different guidesequences, or combinations of these. When multiple different guidesequences are used, a single expression construct may be used to targetCRISPR activity to multiple different, corresponding target sequenceswithin a cell. For example, a single vector may comprise about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guidesequences. In some embodiments, about or more than about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more such guide-sequence-containing vectors may beprovided, and optionally delivered to a cell. In some embodiments, avector comprises a regulatory element operably linked to anenzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein.CRISPR enzyme or CRISPR enzyme mRNA or CRISPR guide RNA or RNA(s) can bedelivered separately; and advantageously at least one of these isdelivered via a particle complex. CRISPR enzyme mRNA can be deliveredprior to the guide RNA to give time for CRISPR enzyme to be expressed.CRISPR enzyme mRNA might be administered 1-12 hours (preferably around2-6 hours) prior to the administration of guide RNA. Alternatively,CRISPR enzyme mRNA and guide RNA can be administered together.Advantageously, a second booster dose of guide RNA can be administered1-12 hours (preferably around 2-6 hours) after the initialadministration of CRISPR enzyme mRNA+guide RNA. Additionaladministrations of CRISPR enzyme mRNA and/or guide RNA might be usefulto achieve the most efficient levels of genome modification.

In one aspect, the invention provides methods for using one or moreelements of a CRISPR system. The CRISPR complex of the inventionprovides an effective means for modifying a target polynucleotide. TheCRISPR complex of the invention has a wide variety of utility includingmodifying (e.g., deleting, inserting, translocating, inactivating,activating) a target polynucleotide in a multiplicity of cell types. Assuch the CRISPR complex of the invention has a broad spectrum ofapplications in, e.g., gene therapy, drug screening, disease diagnosis,and prognosis. An exemplary CRISPR complex comprises a CRISPR enzymecomplexed with a guide sequence hybridized to a target sequence withinthe target polynucleotide. The guide sequence is linked to a tracr matesequence, which in turn hybridizes to a tracr sequence. In oneembodiment, this invention provides a method of cleaving a targetpolynucleotide. The method comprises modifying a target polynucleotideusing a CRISPR complex that binds to the target polynucleotide andeffect cleavage of said target polynucleotide. Typically, the CRISPRcomplex of the invention, when introduced into a cell, creates a break(e.g., a single or a double strand break) in the genome sequence. Forexample, the method can be used to cleave a disease gene in a cell. Thebreak created by the CRISPR complex can be repaired by a repairprocesses such as the error prone non-homologous end joining (NHEJ)pathway or the high fidelity homology-directed repair (HDR). Duringthese repair process, an exogenous polynucleotide template can beintroduced into the genome sequence. In some methods, the HDR process isused modify genome sequence. For example, an exogenous polynucleotidetemplate comprising a sequence to be integrated flanked by an upstreamsequence and a downstream sequence is introduced into a cell. Theupstream and downstream sequences share sequence similarity with eitherside of the site of integration in the chromosome. Where desired, adonor polynucleotide can be DNA, e.g., a DNA plasmid, a bacterialartificial chromosome (BAC), a yeast artificial chromosome (YAC), aviral vector, a linear piece of DNA, a PCR fragment, a naked nucleicacid, or a nucleic acid complexed with a delivery vehicle such as aliposome or poloxamer. The exogenous polynucleotide template comprises asequence to be integrated (e.g., a mutated gene). The sequence forintegration may be a sequence endogenous or exogenous to the cell.Examples of a sequence to be integrated include polynucleotides encodinga protein or a non-coding RNA (e.g., a microRNA). Thus, the sequence forintegration may be operably linked to an appropriate control sequence orsequences. Alternatively, the sequence to be integrated may provide aregulatory function. The upstream and downstream sequences in theexogenous polynucleotide template are selected to promote recombinationbetween the chromosomal sequence of interest and the donorpolynucleotide. The upstream sequence is a nucleic acid sequence thatshares sequence similarity with the genome sequence upstream of thetargeted site for integration. Similarly, the downstream sequence is anucleic acid sequence that shares sequence similarity with thechromosomal sequence downstream of the targeted site of integration. Theupstream and downstream sequences in the exogenous polynucleotidetemplate can have 75%, 80%, 85%, 90%, 95%, or 100% sequence identitywith the targeted genome sequence. Preferably, the upstream anddownstream sequences in the exogenous polynucleotide template have about95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the targetedgenome sequence. In some methods, the upstream and downstream sequencesin the exogenous polynucleotide template have about 99% or 100% sequenceidentity with the targeted genome sequence. An upstream or downstreamsequence may comprise from about 20 bp to about 2500 bp, for example,about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400,or 2500 bp. In some methods, the exemplary upstream or downstreamsequence have about 200 bp to about 2000 bp, about 600 bp to about 1000bp, or more particularly about 700 bp to about 1000 bp. In some methods,the exogenous polynucleotide template may further comprise a marker.Such a marker may make it easy to screen for targeted integrations.Examples of suitable markers include restriction sites, fluorescentproteins, or selectable markers. The exogenous polynucleotide templateof the invention can be constructed using recombinant techniques (see,for example, Sambrook et al., 2001 and Ausubel et al., 1996). In amethod for modifying a target polynucleotide by integrating an exogenouspolynucleotide template, a double stranded break is introduced into thegenome sequence by the CRISPR complex, the break is repaired viahomologous recombination an exogenous polynucleotide template such thatthe template is integrated into the genome. The presence of adouble-stranded break facilitates integration of the template. In otherembodiments, this invention provides a method of modifying expression ofa polynucleotide in a eukaryotic cell. The method comprises increasingor decreasing expression of a target polynucleotide by using a CRISPRcomplex that binds to the polynucleotide. In some methods, a targetpolynucleotide can be inactivated to effect the modification of theexpression in a cell. For example, upon the binding of a CRISPR complexto a target sequence in a cell, the target polynucleotide is inactivatedsuch that the sequence is not transcribed, the coded protein is notproduced, or the sequence does not function as the wild-type sequencedoes. For example, a protein or microRNA coding sequence may beinactivated such that the protein or microRNA or pre-microRNA transcriptis not produced. In some methods, a control sequence can be inactivatedsuch that it no longer functions as a control sequence. As used herein,“control sequence” refers to any nucleic acid sequence that effects thetranscription, translation, or accessibility of a nucleic acid sequence.Examples of a control sequence include, a promoter, a transcriptionterminator, and an enhancer are control sequences. The targetpolynucleotide of a CRISPR complex can be any polynucleotide endogenousor exogenous to the eukaryotic cell. For example, the targetpolynucleotide can be a polynucleotide residing in the nucleus of theeukaryotic cell. The target polynucleotide can be a sequence coding agene product (e.g., a protein) or a non-coding sequence (e.g., aregulatory polynucleotide or a junk DNA). Examples of targetpolynucleotides include a sequence associated with a signalingbiochemical pathway, e.g., a signaling biochemical pathway-associatedgene or polynucleotide. Examples of target polynucleotides include adisease associated gene or polynucleotide. A “disease-associated” geneor polynucleotide refers to any gene or polynucleotide which is yieldingtranscription or translation products at an abnormal level or in anabnormal form in cells derived from a disease-affected tissues comparedwith tissues or cells of a non disease control. It may be a gene thatbecomes expressed at an abnormally high level; it may be a gene thatbecomes expressed at an abnormally low level, where the alteredexpression correlates with the occurrence and/or progression of thedisease. A disease-associated gene also refers to a gene possessingmutation(s) or genetic variation that is directly responsible or is inlinkage disequilibrium with a gene(s) that is responsible for theetiology of a disease. The transcribed or translated products may beknown or unknown, and may be at a normal or abnormal level. The targetpolynucleotide of a CRISPR complex can be any polynucleotide endogenousor exogenous to the eukaryotic cell. For example, the targetpolynucleotide can be a polynucleotide residing in the nucleus of theeukaryotic cell. The target polynucleotide can be a sequence coding agene product (e.g., a protein) or a non-coding sequence (e.g., aregulatory polynucleotide or a junk DNA). Without wishing to be bound bytheory, it is believed that the target sequence should be associatedwith a PAM (protospacer adjacent motif); that is, a short sequencerecognized by the CRISPR complex. The precise sequence and lengthrequirements for the PAM differ depending on the CRISPR enzyme used, butPAMs are typically 2-5 base pair sequences adjacent the protospacer(that is, the target sequence) Examples of PAM sequences are giventhroughout, and the skilled person will be able to identify further PAMsequences for use with a given CRISPR enzyme. In some embodiments, themethod comprises allowing a CRISPR complex to bind to the targetpolynucleotide to effect cleavage of said target polynucleotide therebymodifying the target polynucleotide, wherein the CRISPR complexcomprises a CRISPR enzyme complexed with a guide sequence hybridized toa target sequence within said target polynucleotide, wherein said guidesequence is linked to a tracr mate sequence which in turn hybridizes toa tracr sequence. In one aspect, the invention provides a method ofmodifying expression of a polynucleotide in a eukaryotic cell. In someembodiments, the method comprises allowing a CRISPR complex to bind tothe polynucleotide such that said binding results in increased ordecreased expression of said polynucleotide; wherein the CRISPR complexcomprises a CRISPR enzyme complexed with a guide sequence hybridized toa target sequence within said polynucleotide, wherein said guidesequence is linked to a tracr mate sequence which in turn hybridizes toa tracr sequence. Similar considerations and conditions apply as abovefor methods of modifying a target polynucleotide. In fact, thesesampling, culturing and re-introduction options apply across the aspectsof the present invention. In one aspect, the invention provides formethods of modifying a target polynucleotide in a eukaryotic cell, whichmay be in vivo, ex vivo or in vitro. In some embodiments, the methodcomprises sampling a cell or population of cells from a human ornon-human animal, and modifying the cell or cells. Culturing may occurat any stage ex vivo. The cell or cells may even be re-introduced intothe non-human animal or plant. For re-introduced cells it isparticularly preferred that the cells are stem cells.

Indeed, in some aspects of the invention, the CRISPR complex maycomprise a CRISPR enzyme complexed with a guide sequence hybridized to atarget sequence, wherein said guide sequence may be linked to a tracrmate sequence which in turn may hybridize to a tracr sequence.

The invention relates to the engineering and optimization of systems,methods and compositions used for the control of gene expressioninvolving sequence targeting, such as genome perturbation orgene-editing, that relate to the CRISPR-Cas system and componentsthereof. In advantageous embodiments, the Cas enzyme is Cas9. Anadvantage of the present methods is that the CRISPR system minimizes oravoids off-target binding and its resulting side effects. This isachieved using systems arranged to have a high degree of sequencespecificity for the target DNA.

Self-Inactivating Systems

Once intended alterations have been introduced, such as by editingintended copies of a gene in the genome of a cell, continued CRISPR-Cas9system expression in that cell is no longer necessary. Indeed, sustainedexpression would be undesirable in certain casein case of off-targeteffects at unintended genomic sites, etc. Thus time-limited expressionwould be useful. Inducible expression offers one approach, but inaddition Applicants have engineered a Self-Inactivating CRISPR-Cas9system that relies on the use of a non-coding guide target sequencewithin the CRISPR vector itself. Thus, after expression begins, theCRISPR system will lead to its own destruction, but before destructionis complete it will have time to edit the genomic copies of the targetgene (which, with a normal point mutation in a diploid cell, requires atmost two edits). Simply, the self inactivating CRISPR-Cas systemincludes additional RNA (i.e., guide RNA) that targets the codingsequence for the CRISPR enzyme itself or that targets one or morenon-coding guide target sequences complementary to unique sequencespresent in one or more of the following: (a) within the promoter drivingexpression of the non-coding RNA elements, (b) within the promoterdriving expression of the Cas9 gene, (c) within 100 bp of the ATGtranslational start codon in the Cas9 coding sequence, (d) within theinverted terminal repeat (iTR) of a viral delivery vector, e.g., in anAAV genome.

In embodiments of the invention the terms guide sequence and guide RNAare used interchangeably as in herein cited documents such as WO2014/093622 (PCT/US2013/074667). In general, a guide sequence is anypolynucleotide sequence having sufficient complementarity with a targetpolynucleotide sequence to hybridize with the target sequence and directsequence-specific binding of a CRISPR complex to the target sequence. Insome embodiments, the degree of complementarity between a guide sequenceand its corresponding target sequence, when optimally aligned using asuitable alignment algorithm, is about or more than about 50%, 60%, 75%,80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may bedetermined with the use of any suitable algorithm for aligningsequences, non-limiting example of which include the Smith-Watermanalgorithm, the Needleman-Wunsch algorithm, algorithms based on theBurrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW,Clustal X, BLAT, Novoalign (Novocraft Technologies; available atwww.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (availableat soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). Insome embodiments, a guide sequence is about or more than about 5, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In someembodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30,25, 20, 15, 12, or fewer nucleotides in length. Preferably the guidesequence is 10-30 nucleotides long. The ability of a guide sequence todirect sequence-specific binding of a CRISPR complex to a targetsequence may be assessed by any suitable assay. For example, thecomponents of a CRISPR system sufficient to form a CRISPR complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target sequence, such as by transfectionwith vectors encoding the components of the CRISPR sequence, followed byan assessment of preferential cleavage within the target sequence, suchas by Surveyor assay as described herein (for DNA cleavage) or RT-qPCRanalysis to determine RNA interference. Similarly, cleavage of a targetpolynucleotide sequence may be evaluated in a test tube by providing thetarget sequence, components of a CRISPR complex, including the guidesequence to be tested and a control guide sequence different from thetest guide sequence, and comparing binding or rate of cleavage at thetarget sequence between the test and control guide sequence reactions.Other assays are possible, and will occur to those skilled in the art. Aguide sequence may be selected to target any target sequence. In someembodiments, the target sequence is a sequence within a genome of acell. Exemplary target sequences include those that are unique in thetarget genome.

In an embodiment, a guide RNA molecule can be targeted to a knowntranscription response elements (e.g., promoters, enhancers, etc.), aknown upstream activating sequences, and/or sequences of unknown orknown function that are suspected of being able to control expression ofthe target DNA.

In some methods, a target polynucleotide can be inactivated to effectthe modification of the expression in a cell. For example, upon thebinding of a CRISPR complex to a target sequence in a cell, the targetpolynucleotide is inactivated such that the sequence is not transcribed,the coded protein is not produced, or the sequence does not function asthe wild-type sequence does. For example, a protein or microRNA codingsequence may be inactivated such that the protein is not produced.

In certain embodiments, the CRISPR enzyme comprises one or moremutations selected from the group consisting of D917A, E1006A and D1225Aand/or the one or more mutations is in a RuvC domain of the CRISPRenzyme or is a mutation as otherwise as discussed herein. In someembodiments, the CRISPR enzyme has one or more mutations in a catalyticdomain, wherein when transcribed, the direct repeat sequence forms asingle stem loop and the guide sequence directs sequence-specificbinding of a CRISPR complex to the target sequence, and wherein theenzyme further comprises a functional domain. In some embodiments, thefunctional domain is a transcriptional activation domain, preferablyVP64. In some embodiments, the functional domain is a transcriptionrepression domain, preferably KRAB. In some embodiments, thetranscription repression domain is SID, or concatemers of SID (egSID4X). In some embodiments, the functional domain is an epigeneticmodifying domain, such that an epigenetic modifying enzyme is provided.In some embodiments, the functional domain is an activation domain,which may be the P65 activation domain.

Delivery Generally

Through this disclosure and the knowledge in the art, CRISPR-Cas system,or components thereof or nucleic acid molecules thereof (including, forinstance HDR template) or nucleic acid molecules encoding or providingcomponents thereof may be delivered by a delivery system hereindescribed both generally and in detail.

Vector delivery, e.g., plasmid, viral delivery: The CRISPR enzyme, forinstance a Cas9, and/or any of the present RNAs, for instance a guideRNA, can be delivered using any suitable vector, e.g., plasmid or viralvectors, such as adeno associated virus (AAV), lentivirus, adenovirus orother viral vector types, or combinations thereof. Cas9 and one or moreguide RNAs can be packaged into one or more vectors, e.g., plasmid orviral vectors. In some embodiments, the vector, e.g., plasmid or viralvector is delivered to the tissue of interest by, for example, anintramuscular injection, while other times the delivery is viaintravenous, transdermal, intranasal, oral, mucosal, or other deliverymethods. Such delivery may be either via a single dose, or multipledoses. One skilled in the art understands that the actual dosage to bedelivered herein may vary greatly depending upon a variety of factors,such as the vector choice, the target cell, organism, or tissue, thegeneral condition of the subject to be treated, the degree oftransformation/modification sought, the administration route, theadministration mode, the type of transformation/modification sought,etc.

Such a dosage may further contain, for example, a carrier (water,saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin,dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, apharmaceutically-acceptable carrier (e.g., phosphate-buffered saline), apharmaceutically-acceptable excipient, and/or other compounds known inthe art. The dosage may further contain one or more pharmaceuticallyacceptable salts such as, for example, a mineral acid salt such as ahydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and thesalts of organic acids such as acetates, propionates, malonates,benzoates, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, gels or gelling materials,flavorings, colorants, microspheres, polymers, suspension agents, etc.may also be present herein. In addition, one or more other conventionalpharmaceutical ingredients, such as preservatives, humectants,suspending agents, surfactants, antioxidants, anticaking agents,fillers, chelating agents, coating agents, chemical stabilizers, etc.may also be present, especially if the dosage form is a reconstitutableform. Suitable exemplary ingredients include microcrystalline cellulose,carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol,chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propylgallate, the parabens, ethyl vanillin, glycerin, phenol,parachlorophenol, gelatin, albumin and a combination thereof. A thoroughdiscussion of pharmaceutically acceptable excipients is available inREMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991) which isincorporated by reference herein.

In an embodiment herein the delivery is via an adenovirus, which may beat a single booster dose containing at least 1×10⁵ particles (alsoreferred to as particle units, pu) of adenoviral vector. In anembodiment herein, the dose preferably is at least about 1×10⁶ particles(for example, about 1×10⁶-1×10¹² particles), more preferably at leastabout 1×10⁷ particles, more preferably at least about 1×10⁸ particles(e.g., about 1×10⁸-1×10¹¹ particles or about 1×10⁸-1×10¹² particles),and most preferably at least about 1×10⁰ particles (e.g., about1×10⁹-1×10¹⁰ particles or about 1×10⁹-1×10¹² particles), or even atleast about 1×10¹⁰ particles (e.g., about 1×10¹⁰-1×10¹² particles) ofthe adenoviral vector. Alternatively, the dose comprises no more thanabout 1×10¹⁴ particles, preferably no more than about 1×10¹³ particles,even more preferably no more than about 1×10¹² particles, even morepreferably no more than about 1×10¹¹ particles, and most preferably nomore than about 1×10¹⁰ particles (e.g., no more than about 1×10⁹articles). Thus, the dose may contain a single dose of adenoviral vectorwith, for example, about 1×10⁶ particle units (pu), about 2×10⁶ pu,about 4×10⁶ pu, about 1×10⁷ pu, about 2×10⁷ pu, about 4×10⁷ pu, about1×10⁸ pu, about 2×10⁸ pu, about 4×10⁸ pu, about 1×10⁹ pu, about 2×10⁹pu, about 4×10⁹ pu, about 1×10¹⁰ pu about 2×10¹⁰ pu about 4×10¹⁰ pu,about 1×10¹¹ pu, about 2×10¹¹ pu, about 4×10¹¹ pu, about 1×10¹² pu,about 2×10¹² pu, or about 4×10¹² pu of adenoviral vector. See, forexample, the adenoviral vectors in U.S. Pat. No. 8,454,972 B2 to Nabel,et. al., granted on Jun. 4, 2013; incorporated by reference herein, andthe dosages at col 29, lines 36-58 thereof. In an embodiment herein, theadenovirus is delivered via multiple doses.

In an embodiment herein, the delivery is via an AAV. A therapeuticallyeffective dosage for in vivo delivery of the AAV to a human is believedto be in the range of from about 20 to about 50 ml of saline solutioncontaining from about 1×10¹⁰ to about 1×10¹⁰ functional AAV/ml solution.The dosage may be adjusted to balance the therapeutic benefit againstany side effects. In an embodiment herein, the AAV dose is generally inthe range of concentrations of from about 1×10⁵ to 1×10⁵⁰ genomes AAV,from about 1×10⁸ to 1×10²⁰ genomes AAV, from about 1×10¹⁰ to about1×10¹⁶ genomes, or about 1×10¹¹ to about 1×10¹⁶ genomes AAV. A humandosage may be about 1×10¹³ genomes AAV. Such concentrations may bedelivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50ml, or about 10 to about 25 ml of a carrier solution. Other effectivedosages can be readily established by one of ordinary skill in the artthrough routine trials establishing dose response curves. See, forexample, U.S. Pat. No. 8,404,658 B2 to Hajjar, et al., granted on Mar.26, 2013, at col. 27, lines 45-60.

In an embodiment herein the delivery is via a plasmid. In such plasmidcompositions, the dosage should be a sufficient amount of plasmid toelicit a response. For instance, suitable quantities of plasmid DNA inplasmid compositions can be from about 0.1 to about 2 mg, or from about1 μg to about 10 μg per 70 kg individual. Plasmids of the invention willgenerally comprise (i) a promoter; (ii) a sequence encoding a CRISPRenzyme, operably linked to said promoter; (iii) a selectable marker;(iv) an origin of replication; and (v) a transcription terminatordownstream of and operably linked to (ii). The plasmid can also encodethe RNA components of a CRISPR complex, but one or more of these mayinstead be encoded on a different vector.

The doses herein are based on an average 70 kg individual. The frequencyof administration is within the ambit of the medical or veterinarypractitioner (e.g., physician, veterinarian), or scientist skilled inthe art. It is also noted that mice used in experiments are typicallyabout 20 g and from mice experiments one can scale up to a 70 kgindividual.

In some embodiments the RNA molecules of the invention are delivered inliposome or lipofectin formulations and the like and can be prepared bymethods well known to those skilled in the art. Such methods aredescribed, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and5,580,859, which are herein incorporated by reference. Delivery systemsaimed specifically at the enhanced and improved delivery of siRNA intomammalian cells have been developed, (see, for example, Shen et al FEBSLet. 2003, 539:111-114; Xia et al., Nat. Biotech. 2002, 20:1006-1010;Reich et al., Mol. Vision. 2003, 9: 210-216; Sorensen et al., J. Mol.Biol. 2003, 327: 761-766; Lewis et al., Nat. Gen. 2002, 32: 107-108 andSimeoni et al., NAR 2003, 31, 11: 2717-2724) and may be applied to thepresent invention. siRNA has recently been successfully used forinhibition of gene expression in primates (see for example. Tolentino etal., Retina 24(4):660 which may also be applied to the presentinvention.

Indeed, RNA delivery is a useful method of in vivo delivery. It ispossible to deliver Cas9 and gRNA (and, for instance, HR repairtemplate) into cells using liposomes or particles or nanoparticles. Thusdelivery of the CRISPR enzyme, such as a Cas9 and/or delivery of theRNAs of the invention may be in RNA form and via microvesicles,liposomes or particles or nanoparticles. For example, Cas9 mRNA and gRNAcan be packaged into liposomal particles for delivery in vivo. Liposomaltransfection reagents such as lipofectamine from Life Technologies andother reagents on the market can effectively deliver RNA molecules intothe liver.

Means of delivery of RNA also preferred include delivery of RNA viaparticles (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei, Y.,Bogatyrev, S., Langer, R. and Anderson, D., Lipid-like nanoparticles forsmall interfering RNA delivery to endothelial cells, Advanced FunctionalMaterials, 19: 3112-3118, 2010) or exosomes (Schroeder, A., Levins, C.,Cortez, C., Langer, R., and Anderson, D., Lipid-based nanotherapeuticsfor siRNA delivery, Journal of Internal Medicine, 267: 9-21, 2010, PMID:20059641). Indeed, exosomes have been shown to be particularly useful indelivery siRNA, a system with some parallels to the CRISPR system. Forinstance, El-Andaloussi S, et al. (“Exosome-mediated delivery of siRNAin vitro and in vivo.” Nat Protoc. 2012 December; 7(12):2112-26. doi:10.1038/nprot.2012.131. Epub 2012 Nov. 15.) describe how exosomes arepromising tools for drug delivery across different biological barriersand can be harnessed for delivery of siRNA in vitro and in vivo. Theirapproach is to generate targeted exosomes through transfection of anexpression vector, comprising an exosomal protein fused with a peptideligand. The exosomes are then purify and characterized from transfectedcell supernatant, then RNA is loaded into the exosomes. Delivery oradministration according to the invention can be performed withexosomes, in particular but not limited to the brain. Vitamin E(α-tocopherol) may be conjugated with CRISPR Cas and delivered to thebrain along with high density lipoprotein (HDL), for example in asimilar manner as was done by Uno et al. (HUMAN GENE THERAPY 22:711-719(June 2011)) for delivering short-interfering RNA (siRNA) to the brain.Mice were infused via Osmotic minipumps (model 1007D; Alzet, Cupertino,Calif.) filled with phosphate-buffered saline (PBS) or free TocsiBACE orToc-siBACE/HDL and connected with Brain Infusion Kit 3 (Alzet). Abrain-infusion cannula was placed about 0.5 mm posterior to the bregmaat midline for infusion into the dorsal third ventricle. Uno et al.found that as little as 3 nmol of Toc-siRNA with HDL could induce atarget reduction in comparable degree by the same ICV infusion method. Asimilar dosage of CRISPR Cas conjugated to α-tocopherol andco-administered with HDL targeted to the brain may be contemplated forhumans in the present invention, for example, about 3 nmol to about 3μmol of CRISPR Cas targeted to the brain may be contemplated. Zou et al.((HUMAN GENE THERAPY 22:465-475 (April 2011)) describes a method oflentiviral-mediated delivery of short-hairpin RNAs targeting PKCγ for invivo gene silencing in the spinal cord of rats. Zou et al. administeredabout 10 μl of a recombinant lentivirus having a titer of 1×10⁹transducing units (TU)/ml by an intrathecal catheter. A similar dosageof CRISPR Cas expressed in a lentiviral vector targeted to the brain maybe contemplated for humans in the present invention, for example, about10-50 ml of CRISPR Cas targeted to the brain in a lentivirus having atiter of 1×10⁹ transducing units (TU)/ml may be contemplated.

In terms of local delivery to the brain, this can be achieved in variousways. For instance, material can be delivered intrastriatally e.g. byinjection. Injection can be performed stereotactically via a craniotomy.

Enhancing NHEJ or HR efficiency is also helpful for delivery. It ispreferred that NHEJ efficiency is enhanced by co-expressingend-processing enzymes such as Trex2 (Dumitrache et al. Genetics. 2011August; 188(4): 787-797). It is preferred that HR efficiency isincreased by transiently inhibiting NHEJ machineries such as Ku70 andKu86. HR efficiency can also be increased by co-expressing prokaryoticor eukaryotic homologous recombination enzymes such as RecBCD, RecA.

Packaging and Promoters Generally

Ways to package Cas9 coding nucleic acid molecules, e.g., DNA, intovectors, e.g., viral vectors, to mediate genome modification in vivoinclude:

To achieve NHEJ-mediated gene knockout:

-   -   Single virus vector:        -   Vector containing two or more expression cassettes:        -   Promoter-Cas9 coding nucleic acid molecule-terminator        -   Prom oter-gRNA1-terminator        -   Promoter-gRNA2-terminator        -   Promoter-gRNA(N)-terminator (up to size limit of vector)    -   Double virus vector:        -   Vector 1 containing one expression cassette for driving the            expression of Cas9        -   Promoter-Cas9 coding nucleic acid molecule-terminator        -   Vector 2 containing one more expression cassettes for            driving the expression of one or more guideRNAs        -   Prom oter-gRNA1-terminator        -   Promoter-gRNA(N)-terminator (up to size limit of vector)

To mediate homology-directed repair.

-   -   In addition to the single and double virus vector approaches        described above, an additional vector is used to deliver a        homology-direct repair template.

The promoter used to drive Cas9 coding nucleic acid molecule expressioncan include:

AAV ITR can serve as a promoter: this is advantageous for eliminatingthe need for an additional promoter element (which can take up space inthe vector). The additional space freed up can be used to drive theexpression of additional elements (gRNA, etc.). Also, ITR activity isrelatively weaker, so can be used to reduce potential toxicity due toover expression of Cas9.

For ubiquitous expression, can use promoters: CMV, CAG, CBh, PGK, SV40,Ferritin heavy or light chains, etc.

For brain or other CNS expression, can use promoters: SynapsinI for allneurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT forGABAergic neurons, etc.

For liver expression, can use Albumin promoter.

For lung expression, can use SP-B.

For endothelial cells, can use ICAM.

For hematopoietic cells can use IFNbeta or CD45.

For Osteoblasts can use OG-2.

The promoter used to drive guide RNA can include:

Pol III promoters such as U6 or H1

Use of Pol II promoter and intronic cassettes to express gRNA

Adeno Associated Virus (AAV)

Cas9 and one or more guide RNA can be delivered using adeno associatedvirus (AAV), lentivirus, adenovirus or other plasmid or viral vectortypes, in particular, using formulations and doses from, for example,U.S. Pat. No. 8,454,972 (formulations, doses for adenovirus), U.S. Pat.No. 8,404,658 (formulations, doses for AAV) and U.S. Pat. No. 5,846,946(formulations, doses for DNA plasmids) and from clinical trials andpublications regarding the clinical trials involving lentivirus, AAV andadenovirus. For examples, for AAV, the route of administration,formulation and dose can be as in U.S. Pat. No. 8,454,972 and as inclinical trials involving AAV. For Adenovirus, the route ofadministration, formulation and dose can be as in U.S. Pat. No.8,404,658 and as in clinical trials involving adenovirus. For plasmiddelivery, the route of administration, formulation and dose can be as inU.S. Pat. No. 5,846,946 and as in clinical studies involving plasmids.Doses may be based on or extrapolated to an average 70 kg individual(e.g. a male adult human), and can be adjusted for patients, subjects,mammals of different weight and species. Frequency of administration iswithin the ambit of the medical or veterinary practitioner (e.g.,physician, veterinarian), depending on usual factors including the age,sex, general health, other conditions of the patient or subject and theparticular condition or symptoms being addressed. The viral vectors canbe injected into the tissue of interest. For cell-type specific genomemodification, the expression of Cas9 can be driven by a cell-typespecific promoter. For example, liver-specific expression might use theAlbumin promoter and neuron-specific expression (e.g. for targeting CNSdisorders) might use the Synapsin I promoter.

In terms of in vivo delivery, AAV is advantageous over other viralvectors for a couple of reasons:

-   -   Low toxicity (this may be due to the purification method not        requiring ultra centrifugation of cell particles that can        activate the immune response)    -   Low probability of causing insertional mutagenesis because it        doesn't integrate into the host genome.

AAV has a packaging limit of 4.5 or 4.75 Kb. This means that Cas9 aswell as a promoter and transcription terminator have to be all fit intothe same viral vector. Constructs larger than 4.5 or 4.75 Kb will leadto significantly reduced virus production. SpCas9 is quite large, thegene itself is over 4.1 Kb, which makes it difficult for packing intoAAV. Therefore embodiments of the invention include utilizing homologsof Cas9 that are shorter. For example:

Species Cas9 Size Corynebacter diphtheriae 3252 Eubacterium ventriosum3321 Streptococcus pasteurianus 3390 Lactobacillus farciminis 3378Sphaerochaeta globus 3537 Azospirillum B510 3504 Gluconacetobacterdiazotrophicus 3150 Neisseria cinerea 3246 Roseburia intestinalis 3420Parvibaculum lavamentivorans 3111 Staphylococcus aureus 3159Nitratifractor salsuginis DSM 16511 3396 Campylobacter lari CF89-12 3009Streptococcus thermophilus LMD-9 3396

These species are therefore, in general, preferred Cas9 species.

As to AAV, the AAV can be AAV1, AAV2, AAV5 or any combination thereof.One can select the AAV of the AAV with regard to the cells to betargeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid capsidAAV1, AAV2, AAV5 or any combination thereof for targeting brain orneuronal cells; and one can select AAV4 for targeting cardiac tissue.AAV8 is useful for delivery to the liver. The herein promoters andvectors are preferred individually. A tabulation of certain AAVserotypes as to these cells (see Grimm, D. et al, J. Virol. 82:5887-5911 (2008)) is as follows:

Cell Line AAV-1 AAV-2 AAV-3 AAV-4 AAV-5 AAV-6 AAV-8 AAV-9 Huh-7 13 1002.5 0.0 0.1 10 0.7 0.0 HEK293 25 100 2.5 0.1 0.1 5 0.7 0.1 HeLa 3 1002.0 0.1 6.7 1 0.2 0.1 HepG2 3 100 16.7 0.3 1.7 5 0.3 ND Hep1A 20 100 0.21.0 0.1 1 0.2 0.0 911 17 100 11 0.2 0.1 17 0.1 ND CHO 100 100 14 1.4 33350 10 1.0 COS 33 100 33 3.3 5.0 14 2.0 0.5 MeWo 10 100 20 0.3 6.7 10 1.00.2 NIH3T3 10 100 2.9 2.9 0.3 10 0.3 ND A549 14 100 20 ND 0.5 10 0.5 0.1HT1180 20 100 10 0.1 0.3 33 0.5 0.1 Monocytes 1111 100 ND ND 125 1429 NDND Immature DC 2500 100 ND ND 222 2857 ND ND Mature DC 2222 100 ND ND333 3333 ND ND

Lentivirus

Lentiviruses are complex retroviruses that have the ability to infectand express their genes in both mitotic and post-mitotic cells. The mostcommonly known lentivirus is the human immunodeficiency virus (HIV),which uses the envelope glycoproteins of other viruses to target a broadrange of cell types.

Lentiviruses may be prepared as follows. After cloning pCasES10 (whichcontains a lentiviral transfer plasmid backbone), HEK293FT at lowpassage (p=5) were seeded in a T-75 flask to 50% confluence the daybefore transfection in DMEM with 10% fetal bovine serum and withoutantibiotics. After 20 hours, media was changed to OptiMEM (serum-free)media and transfection was done 4 hours later. Cells were transfectedwith 10 μg of lentiviral transfer plasmid (pCasES10) and the followingpackaging plasmids: 5 μg of pMD2.G (VSV-g pseudotype), and 7.5 ug ofpsPAX2 (gag/pol/rev/tat). Transfection was done in 4 mL OptiMEM with acationic lipid delivery agent (50 uL Lipofectamine 2000 and 100 ul Plusreagent). After 6 hours, the media was changed to antibiotic-free DMEMwith 10% fetal bovine serum. These methods use serum during cellculture, but serum-free methods are preferred.

Lentivirus may be purified as follows. Viral supernatants were harvestedafter 48 hours. Supernatants were first cleared of debris and filteredthrough a 0.45 um low protein binding (PVDF) filter. They were then spunin a ultracentrifuge for 2 hours at 24,000 rpm. Viral pellets wereresuspended in 50 ul of DMEM overnight at 4 C. They were then aliquottedand immediately frozen at −80° C.

In another embodiment, minimal non-primate lentiviral vectors based onthe equine infectious anemia virus (EIAV) are also contemplated,especially for ocular gene therapy (see, e.g., Balagaan, J Gene Med2006; 8: 275-285). In another embodiment, RetinoStat®, an equineinfectious anemia virus-based lentiviral gene therapy vector thatexpresses angiostatic proteins endostatin and angiostatin that isdelivered via a subretinal injection for the treatment of the web formof age-related macular degeneration is also contemplated (see, e.g.,Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)) and thisvector may be modified for the CRISPR-Cas system of the presentinvention.

In another embodiment, self-inactivating lentiviral vectors with ansiRNA targeting a common exon shared by HIV tat/rev, anucleolar-localizing TAR decoy, and an anti-CCR5-specific hammerheadribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) maybe used/and or adapted to the CRISPR-Cas system of the presentinvention. A minimum of 2.5×10⁶ CD34+ cells per kilogram patient weightmay be collected and prestimulated for 16 to 20 hours in X-VIVO 15medium (Lonza) containing 2 μmol/L-glutamine, stem cell factor (100ng/ml), Flt-3 ligand (Flt-3L) (100 ng/ml), and thrombopoietin (10 ng/ml)(CellGenix) at a density of 2×10⁶ cells/ml. Prestimulated cells may betransduced with lentiviral at a multiplicity of infection of 5 for 16 to24 hours in 75-cm² tissue culture flasks coated with fibronectin (25mg/cm²) (RetroNectin, Takara Bio Inc.).

Lentiviral vectors have been disclosed as in the treatment forParkinson's Disease, see, e.g., US Patent Publication No. 20120295960and U.S. Pat. Nos. 7,303,910 and 7,351,585. Lentiviral vectors have alsobeen disclosed for the treatment of ocular diseases, see e.g., US PatentPublication Nos. 20060281180, 20090007284, US20110117189; US20090017543;US20070054961, US20100317109. Lentiviral vectors have also beendisclosed for delivery to the brain, see, e.g., US Patent PublicationNos. US20110293571; US20110293571, US20040013648, US20070025970,US20090111106 and U.S. Pat. No. 7,259,015.

RNA Delivery

RNA delivery: The CRISPR enzyme, for instance a Cas9, and/or any of thepresent RNAs, for instance a guide RNA, can also be delivered in theform of RNA. Cas9 mRNA can be generated using in vitro transcription.For example, Cas9 mRNA can be synthesized using a PCR cassettecontaining the following elements: T7_promoter-kozak sequence(GCCACC)-Cas9-3′ UTR from beta globin-polyA tail (a string of 120 ormore adenines). The cassette can be used for transcription by T7polymerase. Guide RNAs can also be transcribed using in vitrotranscription from a cassette containing T7_promoter-GG-guide RNAsequence.

To enhance expression and reduce possible toxicity, the CRISPRenzyme-coding sequence and/or the guide RNA can be modified to includeone or more modified nucleoside e.g. using pseudo-U or 5-Methyl-C.

mRNA delivery methods are especially promising for liver deliverycurrently.

Much clinical work on RNA delivery has focused on RNAi or antisense, butthese systems can be adapted for delivery of RNA for implementing thepresent invention. References below to RNAi etc. should be readaccordingly.

Particle Delivery Systems and/or Formulations:

Several types of particle delivery systems and/or formulations are knownto be useful in a diverse spectrum of biomedical applications. Ingeneral, a particle is defined as a small object that behaves as a wholeunit with respect to its transport and properties. Particles are furtherclassified according to diameter Coarse particles cover a range between2,500 and 10,000 nanometers. Fine particles are sized between 100 and2,500 nanometers. Ultrafine particles, or nanoparticles, are generallybetween 1 and 100 nanometers in size. The basis of the 100-nm limit isthe fact that novel properties that differentiate particles from thebulk material typically develop at a critical length scale of under 100nm.

As used herein, a particle delivery system/formulation is defined as anybiological delivery system/formulation which includes a particle inaccordance with the present invention. A particle in accordance with thepresent invention is any entity having a greatest dimension (e.g.diameter) of less than 100 microns (μm). In some embodiments, inventiveparticles have a greatest dimension of less than 10 μm. In someembodiments, inventive particles have a greatest dimension of less than2000 nanometers (nm). In some embodiments, inventive particles have agreatest dimension of less than 1000 nanometers (nm). In someembodiments, inventive particles have a greatest dimension of less than900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100nm. Typically, inventive particles have a greatest dimension (e.g.,diameter) of 500 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 250 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 200 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 150 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 100 nm or less. Smaller particles, e.g., having a greatestdimension of 50 nm or less are used in some embodiments of theinvention. In some embodiments, inventive particles have a greatestdimension ranging between 25 nm and 200 nm.

Particle characterization (including e.g., characterizing morphology,dimension, etc.) is done using a variety of different techniques. Commontechniques are electron microscopy (TEM, SEM), atomic force microscopy(AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy(XPS), powder X-ray diffraction (XRD), Fourier transform infraredspectroscopy (FTIR), matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry(MALDI-TOF), ultraviolet-visiblespectroscopy, dual polarisation interferometry and nuclear magneticresonance (NMR). Characterization (dimension measurements) may be madeas to native particles (i.e., preloading) or after loading of the cargo(herein cargo refers to e.g., one or more components of CRISPR-Cassystem e.g., CRISPR enzyme or mRNA or guide RNA, or any combinationthereof, and may include additional carriers and/or excipients) toprovide particles of an optimal size for delivery for any in vitro, exvivo and/or in vivo application of the present invention. In certainpreferred embodiments, particle dimension (e.g., diameter)characterization is based on measurements using dynamic laser scattering(DLS). Mention is made of U.S. Pat. No. 8,709,843; U.S. Pat. No.6,007,845; U.S. Pat. No. 5,855,913; U.S. Pat. No. 5,985,309; U.S. Pat.No. 5,543,158; and the publication by James E. Dahlman and Carmen Barneset al. Nature Nanotechnology (2014) published online 11 May 2014,doi:10.1038/nnano.2014.84, concerning particles, methods of making andusing them and measurements thereof.

Particles delivery systems within the scope of the present invention maybe provided in any form, including but not limited to solid, semi-solid,emulsion, or colloidal particles. As such any of the delivery systemsdescribed herein, including but not limited to, e.g., lipid-basedsystems, liposomes, micelles, microvesicles, exosomes, or gene gun maybe provided as particle delivery systems within the scope of the presentinvention.

Particles

CRISPR enzyme mRNA and guide RNA may be delivered simultaneously usingparticles or lipid envelopes; for instance, CRISPR enzyme and RNA of theinvention, e.g., as a complex, can be delivered via a particle as inDahlman et al., WO2015089419 A2 and documents cited therein, such as 7C1(see, e.g., James E. Dahlman and Carmen Barnes et al. NatureNanotechnology (2014) published online 11 May 2014,doi:10.1038/nnano.2014.84), e.g., delivery particle comprising lipid orlipidoid and hydrophilic polymer, e.g., cationic lipid and hydrophilicpolymer, for instance wherein the the cationic lipid comprises1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) and/or whereinthe hydrophilic polymer comprises ethylene glycol or polyethylene glycol(PEG); and/or wherein the particle further comprises cholesterol (e.g.,particle from formulation 1=DOTAP 100, DMPC 0, PEG 0, Cholesterol 0;formulation number 2=DOTAP 90, DMPC 0, PEG 10, Cholesterol 0;formulation number 3=DOTAP 90, DMPC 0, PEG 5, Cholesterol 5), whereinparticles are formed using an efficient, multistep process whereinfirst, effector protein and RNA are mixed together, e.g., at a 1:1 molarratio, e.g., at room temperature, e.g., for 30 minutes, e.g., insterile, nuclease free 1×PBS; and separately, DOTAP, DMPC, PEG, andcholesterol as applicable for the formulation are dissolved in alcohol,e.g., 100% ethanol; and, the two solutions are mixed together to formparticles containing the complexes).

For example, Su X, Fricke J, Kavanagh D G, Irvine D J (“In vitro and invivo mRNA delivery using lipid-enveloped pH-responsive polymernanoparticles” Mol Pharm. 2011 Jun. 6; 8(3):774-87. doi:10.1021/mp100390w. Epub 2011 Apr. 1) describes biodegradable core-shellstructured particles with a poly(β-amino ester) (PBAE) core enveloped bya phospholipid bilayer shell. These were developed for in vivo mRNAdelivery. The pH-responsive PBAE component was chosen to promoteendosome disruption, while the lipid surface layer was selected tominimize toxicity of the polycation core. Such are, therefore, preferredfor delivering RNA of the present invention.

Use of nanoparticles and particles to modulate immune responses at thesingle cell level falls into three general categories (1) particledirectly attached to or embedded by an immune cell ex vivo, enabling theparticle to deliver “cargo” or a therapeutic upon injection in vivo,thereby releasing the therapeutic directly; (2) leverage the naturalpropensity of particles to target or be scavenged by phagocytic cells invivo; (3) actively target a specific cell in vivo by modulating thelegiands or antibodies on the surface of the particle for specificity.(Irvine, D. J., Hanson, M. C., Rakhra, K., & Tokatlian, T. (2015).Synthetic Nanoparticles for Vaccines and Immunotherapy. ChemicalReviews, 115(19), 11109-11146.)

Generally, gold nanoparticles (which range in size from 10 to 100 nm),are inert particles which are biocompatible and possess opticalproperties which make the use of gold nanoparticles ideal for diagnosticand photothermal applications, such as in the case of applications indeep tissue. However, gold nanoparticles possess challenges whichinclude prolonged renteion in the hepatobiliary system and they arenonbiodegradable, both of which may lead to issues as to toxicity.(Irvine, D. J. et al. (2015)1d.)

In another example, use of amphilic gold nanoparticles, which comprisean approximately 2.3 nm gold core and an amphilic ligand shell, readilyembedded within erthyrocyte membranes, indicating that amphilic goldparticles may serve as a vector for delivering a therapeutic into redblood cells directly or other cells. (Atukorale, P. et al. “Influence ofthe Glycocalyx and Plasma Membrane Composition on Amphiphilic GoldNanoparticle Association with Erythrocytes,” Nanoscale DOI: 10.1039/C5NR01355K (2015).)

In one embodiment, particles based on self assembling bioadhesivepolymers are contemplated, which may be applied to oral delivery ofpeptides, intravenous delivery of peptides and nasal delivery ofpeptides, all to the brain. Other embodiments, such as oral absorptionand ocular delivery of hydrophobic drugs are also contemplated. Themolecular envelope technology involves an engineered polymer envelopewhich is protected and delivered to the site of the disease (see, e.g.,Mazza, M. et al. ACS Nano, 2013. 7(2): 1016-1026; Siew, A., et al. MolPharm, 2012. 9(1):14-28; Lalatsa, A., et al. J Contr Rel, 2012.161(2):523-36; Lalatsa, A., et al., Mol Pharm, 2012. 9(6):1665-80;Lalatsa, A., et al. Mol Pharm, 2012. 9(6):1764-74; Garrett, N. L., etal. J Biophotonics, 2012. 5(5-6):458-68; Garrett, N. L., et al. J RamanSpect, 2012. 43(5):681-688; Ahmad, S., et al. J Royal Soc Interface2010. 7:S423-33; Uchegbu, I. F. Expert Opin Drug Deliv, 2006.3(5):629-40; Qu, X., et al. Biomacromolecules, 2006. 7(12):3452-9 andUchegbu, I. F., et al. Int J Pharm, 2001. 224:185-199). Doses of about 5mg/kg are contemplated, with single or multiple doses, depending on thetarget tissue.

In one embodiment, particles that can deliver RNA to a cancer cell tostop tumor growth developed by Dan Anderson's lab at MIT may be used/andor adapted to the CRISPR Cas system of the present invention. Inparticular, the Anderson lab developed fully automated, combinatorialsystems for the synthesis, purification, characterization, andformulation of new biomaterials and nanoformulations. See, e.g., Alabiet al., Proc Natl Acad Sci USA. 2013 Aug. 6; 110(32):12881-6; Zhang etal., Adv Mater. 2013 Sep. 6; 25(33):4641-5; Jiang et al., Nano Lett.2013 Mar. 13; 13(3):1059-64; Karagiannis et al., ACS Nano. 2012 Oct. 23;6(10):8484-7; Whitehead et al., ACS Nano. 2012 Aug. 28; 6(8):6922-9 andLee et al., Nat Nanotechnol. 2012 Jun. 3; 7(6):389-93.

U.S. patent application 20110293703 relates to lipidoid compounds arealso particularly useful in the administration of polynucleotides, whichmay be applied to deliver the CRISPR Cas system of the presentinvention. In one aspect, the aminoalcohol lipidoid compounds arecombined with an agent to be delivered to a cell or a subject to formmicroparticles, nanoparticles, liposomes, or micelles. The agent to bedelivered by the particles, liposomes, or micelles may be in the form ofa gas, liquid, or solid, and the agent may be a polynucleotide, protein,peptide, or small molecule. The minoalcohol lipidoid compounds may becombined with other aminoalcohol lipidoid compounds, polymers (syntheticor natural), surfactants, cholesterol, carbohydrates, proteins, lipids,etc. to form the particles. These particles may then optionally becombined with a pharmaceutical excipient to form a pharmaceuticalcomposition.

US Patent Publication No. 20110293703 also provides methods of preparingthe aminoalcohol lipidoid compounds. One or more equivalents of an amineare allowed to react with one or more equivalents of anepoxide-terminated compound under suitable conditions to form anaminoalcohol lipidoid compound of the present invention. In certainembodiments, all the amino groups of the amine are fully reacted withthe epoxide-terminated compound to form tertiary amines. In otherembodiments, all the amino groups of the amine are not fully reactedwith the epoxide-terminated compound to form tertiary amines therebyresulting in primary or secondary amines in the aminoalcohol lipidoidcompound. These primary or secondary amines are left as is or may bereacted with another electrophile such as a different epoxide-terminatedcompound. As will be appreciated by one skilled in the art, reacting anamine with less than excess of epoxide-terminated compound will resultin a plurality of different aminoalcohol lipidoid compounds with variousnumbers of tails. Certain amines may be fully functionalized with twoepoxide-derived compound tails while other molecules will not becompletely functionalized with epoxide-derived compound tails. Forexample, a diamine or polyamine may include one, two, three, or fourepoxide-derived compound tails off the various amino moieties of themolecule resulting in primary, secondary, and tertiary amines. Incertain embodiments, all the amino groups are not fully functionalized.In certain embodiments, two of the same types of epoxide-terminatedcompounds are used. In other embodiments, two or more differentepoxide-terminated compounds are used. The synthesis of the aminoalcohollipidoid compounds is performed with or without solvent, and thesynthesis may be performed at higher temperatures ranging from 30-100°C., preferably at approximately 50-90° C. The prepared aminoalcohollipidoid compounds may be optionally purified. For example, the mixtureof aminoalcohol lipidoid compounds may be purified to yield anaminoalcohol lipidoid compound with a particular number ofepoxide-derived compound tails. Or the mixture may be purified to yielda particular stereo- or regioisomer. The aminoalcohol lipidoid compoundsmay also be alkylated using an alkyl halide (e.g., methyl iodide) orother alkylating agent, and/or they may be acylated.

US Patent Publication No. 20110293703 also provides libraries ofaminoalcohol lipidoid compounds prepared by the inventive methods. Theseaminoalcohol lipidoid compounds may be prepared and/or screened usinghigh-throughput techniques involving liquid handlers, robots, microtiterplates, computers, etc. In certain embodiments, the aminoalcohollipidoid compounds are screened for their ability to transfectpolynucleotides or other agents (e.g., proteins, peptides, smallmolecules) into the cell.

US Patent Publication No. 20130302401 relates to a class ofpoly(beta-amino alcohols) (PBAAs) has been prepared using combinatorialpolymerization. The inventive PBAAs may be used in biotechnology andbiomedical applications as coatings (such as coatings of films ormultilayer films for medical devices or implants), additives, materials,excipients, non-biofouling agents, micropatterning agents, and cellularencapsulation agents. When used as surface coatings, these PBAAselicited different levels of inflammation, both in vitro and in vivo,depending on their chemical structures. The large chemical diversity ofthis class of materials allowed us to identify polymer coatings thatinhibit macrophage activation in vitro. Furthermore, these coatingsreduce the recruitment of inflammatory cells, and reduce fibrosis,following the subcutaneous implantation of carboxylated polystyrenemicroparticles. These polymers may be used to form polyelectrolytecomplex capsules for cell encapsulation. The invention may also havemany other biological applications such as antimicrobial coatings, DNAor siRNA delivery, and stem cell tissue engineering. The teachings of USPatent Publication No. 20130302401 may be applied to the CRISPR Cassystem of the present invention.

In another embodiment, lipid particles (LNPs) are contemplated. Anantitransthyretin small interfering RNA has been encapsulated in lipidparticles and delivered to humans (see, e.g., Coelho et al., N Engl JMed 2013; 369:819-29), and such a system may be adapted and applied tothe CRISPR Cas system of the present invention. Doses of about 0.01 toabout 1 mg per kg of body weight administered intravenously arecontemplated. Medications to reduce the risk of infusion-relatedreactions are contemplated, such as dexamethasone, acetampinophen,diphenhydramine or cetirizine, and ranitidine are contemplated. Multipledoses of about 0.3 mg per kilogram every 4 weeks for five doses are alsocontemplated.

LNPs have been shown to be highly effective in delivering siRNAs to theliver (see, e.g., Tabernero et al., Cancer Discovery, April 2013, Vol.3, No. 4, pages 363-470) and are therefore contemplated for deliveringRNA encoding CRISPR Cas to the liver. A dosage of about four doses of 6mg/kg of the LNP every two weeks may be contemplated. Tabernero et al.demonstrated that tumor regression was observed after the first 2 cyclesof LNPs dosed at 0.7 mg/kg, and by the end of 6 cycles the patient hadachieved a partial response with complete regression of the lymph nodemetastasis and substantial shrinkage of the liver tumors. A completeresponse was obtained after 40 doses in this patient, who has remainedin remission and completed treatment after receiving doses over 26months. Two patients with RCC and extrahepatic sites of diseaseincluding kidney, lung, and lymph nodes that were progressing followingprior therapy with VEGF pathway inhibitors had stable disease at allsites for approximately 8 to 12 months, and a patient with PNET andliver metastases continued on the extension study for 18 months (36doses) with stable disease.

However, the charge of the LNP must be taken into consideration. Ascationic lipids combined with negatively charged lipids to inducenonbilayer structures that facilitate intracellular delivery. Becausecharged LNPs are rapidly cleared from circulation following intravenousinjection, ionizable cationic lipids with pKa values below 7 weredeveloped (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12,pages 1286-2200, December 2011). Negatively charged polymers such as RNAmay be loaded into LNPs at low pH values (e.g., pH 4) where theionizable lipids display a positive charge. However, at physiological pHvalues, the LNPs exhibit a low surface charge compatible with longercirculation times. Four species of ionizable cationic lipids have beenfocused upon, namely 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and1,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane (DLinKC2-DMA). Ithas been shown that LNP siRNA systems containing these lipids exhibitremarkably different gene silencing properties in hepatocytes in vivo,with potencies varying according to the seriesDLinKC2-DMA>DLinKDMA>DLinDMA>>DLinDAP employing a Factor VII genesilencing model (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no.12, pages 1286-2200, December 2011). A dosage of 1 μg/ml of LNP orCRISPR-Cas RNA in or associated with the LNP may be contemplated,especially for a formulation containing DLinKC2-DMA.

Preparation of LNPs and CRISPR Cas encapsulation may be used/and oradapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages1286-2200, December 2011). The cationic lipids1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA),1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA),(3-o[2″-(methoxypolyethyleneglycol 2000)succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), andR-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) may be providedby Tekmira Pharmaceuticals (Vancouver, Canada) or synthesized.Cholesterol may be purchased from Sigma (St Louis, Mo.). The specificCRISPR Cas RNA may be encapsulated in LNPs containing DLinDAP, DLinDMA,DLinK-DMA, and DLinKC2-DMA (cationic lipid:DSPC:CHOL:PEGS-DMG orPEG-C-DOMG at 40:10:40:10 molar ratios). When required, 0.2% SP-DiOC18(Invitrogen, Burlington, Canada) may be incorporated to assess cellularuptake, intracellular delivery, and biodistribution. Encapsulation maybe performed by dissolving lipid mixtures comprised of cationiclipid:DSPC:cholesterol:PEG-c-DOMG (40:10:40:10 molar ratio) in ethanolto a final lipid concentration of 10 mmol/1. This ethanol solution oflipid may be added drop-wise to 50 mmol/1 citrate, pH 4.0 to formmultilamellar vesicles to produce a final concentration of 30% ethanolvol/vol. Large unilamellar vesicles may be formed following extrusion ofmultilamellar vesicles through two stacked 80 nm Nuclepore polycarbonatefilters using the Extruder (Northern Lipids, Vancouver, Canada).Encapsulation may be achieved by adding RNA dissolved at 2 mg/ml in 50mmol/1 citrate, pH 4.0 containing 30% ethanol vol/vol drop-wise toextruded preformed large unilamellar vesicles and incubation at 31° C.for 30 minutes with constant mixing to a final RNA/lipid weight ratio of0.06/1 wt/wt. Removal of ethanol and neutralization of formulationbuffer were performed by dialysis against phosphate-buffered saline(PBS), pH 7.4 for 16 hours using Spectra/Por 2 regenerated cellulosedialysis membranes. Particle size distribution may be determined bydynamic light scattering using a NICOMP 370 particle sizer, thevesicle/intensity modes, and Gaussian fitting (Nicomp Particle Sizing,Santa Barbara, Calif.). The particle size for all three LNP systems maybe ˜70 nm in diameter. RNA encapsulation efficiency may be determined byremoval of free RNA using VivaPureD MiniH columns (Sartorius StedimBiotech) from samples collected before and after dialysis. Theencapsulated RNA may be extracted from the eluted particles andquantified at 260 nm. RNA to lipid ratio was determined by measurementof cholesterol content in vesicles using the Cholesterol E enzymaticassay from Wako Chemicals USA (Richmond, Va.). In conjunction with theherein discussion of LNPs and PEG lipids, PEGylated liposomes or LNPsare likewise suitable for delivery of a CRISPR-Cas system or componentsthereof.

Preparation of large LNPs may be used/and or adapted from Rosin et al,Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December 2011. Alipid premix solution (20.4 mg/ml total lipid concentration) may beprepared in ethanol containing DLinKC2-DMA, DSPC, and cholesterol at50:10:38.5 molar ratios. Sodium acetate may be added to the lipid premixat a molar ratio of 0.75:1 (sodium acetate:DLinKC2-DMA). The lipids maybe subsequently hydrated by combining the mixture with 1.85 volumes ofcitrate buffer (10 mmol/1, pH 3.0) with vigorous stirring, resulting inspontaneous liposome formation in aqueous buffer containing 35% ethanol.The liposome solution may be incubated at 37° C. to allow fortime-dependent increase in particle size. Aliquots may be removed atvarious times during incubation to investigate changes in liposome sizeby dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments,Worcestershire, UK). Once the desired particle size is achieved, anaqueous PEG lipid solution (stock=10 mg/ml PEG-DMG in 35% (vol/vol)ethanol) may be added to the liposome mixture to yield a final PEG molarconcentration of 3.5% of total lipid. Upon addition of PEG-lipids, theliposomes should their size, effectively quenching further growth. RNAmay then be added to the empty liposomes at an RNA to total lipid ratioof approximately 1:10 (wt:wt), followed by incubation for 30 minutes at37° C. to form loaded LNPs. The mixture may be subsequently dialyzedovernight in PBS and filtered with a 0.45-μm syringe filter.

Spherical Nucleic Acid (SNA™) constructs and other particles(particularly gold particles) are also contemplated as a means todelivery CRISPR-Cas system to intended targets. Significant data showthat AuraSense Therapeutics' Spherical Nucleic Acid (SNA™) constructs,based upon nucleic acid-functionalized gold particles, are useful.

Literature that may be employed in conjunction with herein teachingsinclude: Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257, Hao etal., Small. 2011 7:3158-3162, Zhang et al., ACS Nano. 2011 5:6962-6970,Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391, Young et al., NanoLett. 2012 12:3867-71, Zheng et al., Proc. Natl. Acad. Sci. USA. 2012109:11975-80, Mirkin, Nanomedicine 2012 7:635-638 Zhang et al., J. Am.Chem. Soc. 2012 134:16488-1691, Weintraub, Nature 2013 495:S14-S16, Choiet al., Proc. Natl. Acad. Sci. USA. 2013 110(19):7625-7630, Jensen etal., Sci. Transl. Med. 5, 209ra152 (2013) and Mirkin, et al., Small,10:186-192.

Self-assembling particles with RNA may be constructed withpolyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD)peptide ligand attached at the distal end of the polyethylene glycol(PEG). This system has been used, for example, as a means to targettumor neovasculature expressing integrins and deliver siRNA inhibitingvascular endothelial growth factor receptor-2 (VEGF R2) expression andthereby achieve tumor angiogenesis (see, e.g., Schiffelers et al.,Nucleic Acids Research, 2004, Vol. 32, No. 19). Nanoplexes may beprepared by mixing equal volumes of aqueous solutions of cationicpolymer and nucleic acid to give a net molar excess of ionizablenitrogen (polymer) to phosphate (nucleic acid) over the range of 2 to 6.The electrostatic interactions between cationic polymers and nucleicacid resulted in the formation of polyplexes with average particle sizedistribution of about 100 nm, hence referred to here as nanoplexes. Adosage of about 100 to 200 mg of CRISPR Cas is envisioned for deliveryin the self-assembling particles of Schiffelers et al.

The nanoplexes of Bartlett et al. (PNAS, Sep. 25, 2007,vol. 104, no. 39)may also be applied to the present invention. The nanoplexes of Bartlettet al. are prepared by mixing equal volumes of aqueous solutions ofcationic polymer and nucleic acid to give a net molar excess ofionizable nitrogen (polymer) to phosphate (nucleic acid) over the rangeof 2 to 6. The electrostatic interactions between cationic polymers andnucleic acid resulted in the formation of polyplexes with averageparticle size distribution of about 100 nm, hence referred to here asnanoplexes. The DOTA-siRNA of Bartlett et al. was synthesized asfollows: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acidmono(N-hydroxysuccinimide ester) (DOTA-NHSester) was ordered fromMacrocyclics (Dallas, Tex.). The amine modified RNA sense strand with a100-fold molar excess of DOTA-NHS-ester in carbonate buffer (pH 9) wasadded to a microcentrifuge tube. The contents were reacted by stirringfor 4 h at room temperature. The DOTA-RNAsense conjugate wasethanol-precipitated, resuspended in water, and annealed to theunmodified antisense strand to yield DOTA-siRNA. All liquids werepretreated with Chelex-100 (Bio-Rad, Hercules, Calif.) to remove tracemetal contaminants. Tf-targeted and nontargeted siRNA particles may beformed by using cyclodextrin-containing polycations. Typically,particles were formed in water at a charge ratio of 3 (+/−) and an siRNAconcentration of 0.5 g/liter. One percent of the adamantane-PEGmolecules on the surface of the targeted particles were modified with Tf(adamantane-PEG-Tf). The particles were suspended in a 5% (wt/vol)glucose carrier solution for injection.

Davis et al. (Nature, Vol 464, 15 Apr. 2010) conducts a RNA clinicaltrial that uses a targeted particle-delivery system (clinical trialregistration number NCT00689065). Patients with solid cancers refractoryto standard-of-care therapies are administered doses of targetedparticles on days 1, 3, 8 and 10 of a 21-day cycle by a 30-minintravenous infusion. The particles consist of a synthetic deliverysystem containing: (1) a linear, cyclodextrin-based polymer (CDP), (2) ahuman transferrin protein (TF) targeting ligand displayed on theexterior of the particle to engage TF receptors (TFR) on the surface ofthe cancer cells, (3) a hydrophilic polymer (polyethylene glycol (PEG)used to promote particle stability in biological fluids), and (4) siRNAdesigned to reduce the expression of the RRM2 (sequence used in theclinic was previously denoted siR2B+5). The TFR has long been known tobe upregulated in malignant cells, and RRM2 is an establishedanti-cancer target. These particles (clinical version denoted asCALAA-01) have been shown to be well tolerated in multi-dosing studiesin non-human primates. Although a single patient with chronic myeloidleukaemia has been administered siRNAby liposomal delivery, Davis etal.'s clinical trial is the initial human trial to systemically deliversiRNA with a targeted delivery system and to treat patients with solidcancer. To ascertain whether the targeted delivery system can provideeffective delivery of functional siRNA to human tumours, Davis et al.investigated biopsies from three patients from three different dosingcohorts; patients A, B and C, all of whom had metastatic melanoma andreceived CALAA-01 doses of 18, 24 and 30 mg m⁻² siRNA, respectively.Similar doses may also be contemplated for the CRISPR Cas system of thepresent invention. The delivery of the invention may be achieved withparticles containing a linear, cyclodextrin-based polymer (CDP), a humantransferrin protein (TF) targeting ligand displayed on the exterior ofthe particle to engage TF receptors (TFR) on the surface of the cancercells and/or a hydrophilic polymer (for example, polyethylene glycol(PEG) used to promote particle stability in biological fluids).

In terms of this invention, it is preferred to have one or morecomponents of CRISPR complex, e.g., CRISPR enzyme or mRNA or guide RNAor sgRNA or if present HDR template may be delivered using one or moreparticles or particles or lipid envelopes. Other delivery systems orvectors are may be used in conjunction with the particle aspects of theinvention.

In general, a “nanoparticle” refers to any particle having a diameter ofless than 1000 nm. In certain preferred embodiments, nanoparticles ofthe invention have a greatest dimension (e.g., diameter) of 500 nm orless. In other preferred embodiments, nanoparticles of the inventionhave a greatest dimension ranging between 25 nm and 200 nm. In otherpreferred embodiments, nanoparticles of the invention have a greatestdimension of 100 nm or less. In other preferred embodiments,nanoparticles of the invention have a greatest dimension ranging between35 nm and 60 nm.

Particles encompassed in the present invention may be provided indifferent forms, e.g., as solid particles (e.g., metal such as silver,gold, iron, titanium), non-metal, lipid-based solids, polymers),suspensions of particles, or combinations thereof. Metal, dielectric,and semiconductor particles may be prepared, as well as hybridstructures (e.g., core-shell particles). Particles made ofsemiconducting material may also be labeled quantum dots if they aresmall enough (typically sub 10 nm) that quantization of electronicenergy levels occurs. Such nanoscale particles are used in biomedicalapplications as drug carriers or imaging agents and may be adapted forsimilar purposes in the present invention.

Semi-solid and soft particles have been manufactured, and are within thescope of the present invention. A prototype particle of semi-solidnature is the liposome. Various types of liposome particles arecurrently used clinically as delivery systems for anticancer drugs andvaccines. Particles with one half hydrophilic and the other halfhydrophobic are termed Janus particles and are particularly effectivefor stabilizing emulsions. They can self-assemble at water/oilinterfaces and act as solid surfactants.

U.S. Pat. No. 8,709,843, incorporated herein by reference, provides adrug delivery system for targeted delivery of therapeuticagent-containing particles to tissues, cells, and intracellularcompartments. The invention provides targeted particles comprisingcomprising polymer conjugated to a surfactant, hydrophilic polymer orlipid. U.S. Pat. No. 6,007,845, incorporated herein by reference,provides particles which have a core of a multiblock copolymer formed bycovalently linking a multifunctional compound with one or morehydrophobic polymers and one or more hydrophilic polymers, and contain abiologically active material. U.S. Pat. No. 5,855,913, incorporatedherein by reference, provides a particulate composition havingaerodynamically light particles having a tap density of less than 0.4g/cm3 with a mean diameter of between 5 μm and 30 μm, incorporating asurfactant on the surface thereof for drug delivery to the pulmonarysystem. U.S. Pat. No. 5,985,309, incorporated herein by reference,provides particles incorporating a surfactant and/or a hydrophilic orhydrophobic complex of a positively or negatively charged therapeutic ordiagnostic agent and a charged molecule of opposite charge for deliveryto the pulmonary system. U.S. Pat. No. 5,543,158, incorporated herein byreference, provides biodegradable injectable particles having abiodegradable solid core containing a biologically active material andpoly(alkylene glycol) moieties on the surface. WO2012135025 (alsopublished as US20120251560), incorporated herein by reference, describesconjugated polyethyleneimine (PEI) polymers and conjugatedaza-macrocycles (collectively referred to as “conjugated lipomer” or“lipomers”). In certain embodiments, it can be envisioned that suchmethods and materials of herein-cited documents, e.g., conjugatedlipomers, can be used in the context of the CRISPR-Cas system to achievein vitro, ex vivo and in vivo genomic perturbations to modify geneexpression, including modulation of protein expression.

In one embodiment, the particle may be epoxide-modified lipid-polymer,advantageously 7C1 (see, e.g., James E. Dahlman and Carmen Barnes et al.Nature Nanotechnology (2014) published online 11 May 2014,doi:10.1038/nnano.2014.84). C71 was synthesized by reacting C15epoxide-terminated lipids with PEI600 at a 14:1 molar ratio, and wasformulated with C14PEG2000 to produce particles (diameter between 35 and60 nm) that were stable in PBS solution for at least 40 days. Anepoxide-modified lipid-polymer may be utilized to deliver the CRISPR-Cassystem of the present invention to pulmonary, cardiovascular or renalcells, however, one of skill in the art may adapt the system to deliverto other target organs. Dosage ranging from about 0.05 to about 0.6mg/kg are envisioned. Dosages over several days or weeks are alsoenvisioned, with a total dosage of about 2 mg/kg.

Exosomes

Exosomes are endogenous nano-vesicles that transport RNAs and proteins,and which can deliver RNA to the brain and other target organs. Toreduce immunogenicity, Alvarez-Erviti et al. (2011, Nat Biotechnol 29:341) used self-derived dendritic cells for exosome production. Targetingto the brain was achieved by engineering the dendritic cells to expressLamp2b, an exosomal membrane protein, fused to the neuron-specific RVGpeptide. Purified exosomes were loaded with exogenous RNA byelectroporation. Intravenously injected RVG-targeted exosomes deliveredGAPDH siRNA specifically to neurons, microglia, oligodendrocytes in thebrain, resulting in a specific gene knockdown. Pre-exposure to RVGexosomes did not attenuate knockdown, and non-specific uptake in othertissues was not observed. The therapeutic potential of exosome-mediatedsiRNA delivery was demonstrated by the strong mRNA (60%) and protein(62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease.

To obtain a pool of immunologically inert exosomes, Alvarez-Erviti etal. harvested bone marrow from inbred C57BL/6 mice with a homogenousmajor histocompatibility complex (MHC) haplotype. As immature dendriticcells produce large quantities of exosomes devoid of T-cell activatorssuch as MHC-II and CD86, Alvarez-Erviti et al. selected for dendriticcells with granulocyte/macrophage-colony stimulating factor (GM-CSF) for7 d. Exosomes were purified from the culture supernatant the followingday using well-established ultracentrifugation protocols. The exosomesproduced were physically homogenous, with a size distribution peaking at80 nm in diameter as determined by nanoparticle tracking analysis (NTA)and electron microscopy. Alvarez-Erviti et al. obtained 6-12 μg ofexosomes (measured based on protein concentration) per 10⁶ cells.

Next, Alvarez-Erviti et al. investigated the possibility of loadingmodified exosomes with exogenous cargoes using electroporation protocolsadapted for nanoscale applications. As electroporation for membraneparticles at the nanometer scale is not well-characterized, nonspecificCy5-labeled RNA was used for the empirical optimization of theelectroporation protocol. The amount of encapsulated RNA was assayedafter ultracentrifugation and lysis of exosomes. Electroporation at 400V and 125 μF resulted in the greatest retention of RNA and was used forall subsequent experiments.

Alvarez-Erviti et al. administered 150 μg of each BACE1 siRNAencapsulated in 150 μg of RVG exosomes to normal C57BL/6 mice andcompared the knockdown efficiency to four controls: untreated mice, miceinjected with RVG exosomes only, mice injected with BACE1 siRNAcomplexed to an in vivo cationic liposome reagent and mice injected withBACE1 siRNA complexed to RVG-9R, the RVG peptide conjugated to 9D-arginines that electrostatically binds to the siRNA. Cortical tissuesamples were analyzed 3 d after administration and a significant proteinknockdown (45%, P<0.05, versus 62%, P<0.01) in both siRNA-RVG-9R-treatedand siRNARVG exosome-treated mice was observed, resulting from asignificant decrease in BACE1 mRNA levels (66% [+ or −] 15%, P<0.001 and61% [+ or −] 13% respectively, P<0.01). Moreover, Applicantsdemonstrated a significant decrease (55%, P<0.05) in the total[beta]-amyloid 1-42 levels, a main component of the amyloid plaques inAlzheimer's pathology, in the RVG-exosome-treated animals. The decreaseobserved was greater than the β-amyloid 1-40 decrease demonstrated innormal mice after intraventricular injection of BACE1 inhibitors.Alvarez-Erviti et al. carried out 5′-rapid amplification of cDNA ends(RACE) on BACE1 cleavage product, which provided evidence ofRNAi-mediated knockdown by the siRNA.

Finally, Alvarez-Erviti et al. investigated whether RNA-RVG exosomesinduced immune responses in vivo by assessing IL-6, IP-10, TNFα andIFN-α serum concentrations. Following exosome treatment, nonsignificantchanges in all cytokines were registered similar to siRNA-transfectionreagent treatment in contrast to siRNA-RVG-9R, which potently stimulatedIL-6 secretion, confirming the immunologically inert profile of theexosome treatment. Given that exosomes encapsulate only 20% of siRNA,delivery with RVG-exosome appears to be more efficient than RVG-9Rdelivery as comparable mRNA knockdown and greater protein knockdown wasachieved with fivefold less siRNA without the corresponding level ofimmune stimulation. This experiment demonstrated the therapeuticpotential of RVG-exosome technology, which is potentially suited forlong-term silencing of genes related to neurodegenerative diseases. Theexosome delivery system of Alvarez-Erviti et al. may be applied todeliver the CRISPR-Cas system of the present invention to therapeutictargets, especially neurodegenerative diseases. A dosage of about 100 to1000 mg of CRISPR Cas encapsulated in about 100 to 1000 mg of RVGexosomes may be contemplated for the present invention.

El-Andaloussi et al. (Nature Protocols 7,2112-2126(2012)) discloses howexosomes derived from cultured cells can be harnessed for delivery ofRNA in vitro and in vivo. This protocol first describes the generationof targeted exosomes through transfection of an expression vector,comprising an exosomal protein fused with a peptide ligand. Next,El-Andaloussi et al. explain how to purify and characterize exosomesfrom transfected cell supernatant. Next, El-Andaloussi et al. detailcrucial steps for loading RNA into exosomes. Finally, El-Andaloussi etal. outline how to use exosomes to efficiently deliver RNA in vitro andin vivo in mouse brain. Examples of anticipated results in whichexosome-mediated RNA delivery is evaluated by functional assays andimaging are also provided. The entire protocol takes ˜3 weeks. Deliveryor administration according to the invention may be performed usingexosomes produced from self-derived dendritic cells. From the hereinteachings, this can be employed in the practice of the invention.

In another embodiment, the plasma exosomes of Wahlgren et al. (NucleicAcids Research, 2012, Vol. 40, No. 17 e130) are contemplated. Exosomesare nano-sized vesicles (30-90 nm in size) produced by many cell types,including dendritic cells (DC), B cells, T cells, mast cells, epithelialcells and tumor cells. These vesicles are formed by inward budding oflate endosomes and are then released to the extracellular environmentupon fusion with the plasma membrane. Because exosomes naturally carryRNA between cells, this property may be useful in gene therapy, and fromthis disclosure can be employed in the practice of the instantinvention.

Exosomes from plasma can be prepared by centrifugation of buffy coat at900 g for 20 min to isolate the plasma followed by harvesting cellsupernatants, centrifuging at 300 g for 10 min to eliminate cells and at16 500 g for 30 min followed by filtration through a 0.22 mm filter.Exosomes are pelleted by ultracentrifugation at 120 000 g for 70 min.Chemical transfection of siRNA into exosomes is carried out according tothe manufacturer's instructions in RNAi Human/Mouse Starter Kit(Quiagen, Hilden, Germany). siRNA is added to 100 ml PBS at a finalconcentration of 2 mmol/ml. After adding HiPerFect transfection reagent,the mixture is incubated for 10 min at RT. In order to remove the excessof micelles, the exosomes are re-isolated using aldehyde/sulfate latexbeads. The chemical transfection of CRISPR Cas into exosomes may beconducted similarly to siRNA. The exosomes may be co-cultured withmonocytes and lymphocytes isolated from the peripheral blood of healthydonors. Therefore, it may be contemplated that exosomes containingCRISPR Cas may be introduced to monocytes and lymphocytes of andautologously reintroduced into a human. Accordingly, delivery oradministration according to the invention may be performed using plasmaexosomes.

Liposomes

Delivery or administration according to the invention can be performedwith liposomes. Liposomes are spherical vesicle structures composed of auni- or multilamellar lipid bilayer surrounding internal aqueouscompartments and a relatively impermeable outer lipophilic phospholipidbilayer. Liposomes have gained considerable attention as drug deliverycarriers because they are biocompatible, nontoxic, can deliver bothhydrophilic and lipophilic drug molecules, protect their cargo fromdegradation by plasma enzymes, and transport their load acrossbiological membranes and the blood brain barrier (BBB) (see, e.g., Spuchand Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12pages, 2011. doi:10.1155/2011/469679 for review).

Liposomes can be made from several different types of lipids; however,phospholipids are most commonly used to generate liposomes as drugcarriers. Although liposome formation is spontaneous when a lipid filmis mixed with an aqueous solution, it can also be expedited by applyingforce in the form of shaking by using a homogenizer, sonicator, or anextrusion apparatus (see, e.g., Spuch and Navarro, Journal of DrugDelivery, vol. 2011, Article ID 469679, 12 pages, 2011.doi:10.1155/2011/469679 for review).

Several other additives may be added to liposomes in order to modifytheir structure and properties. For instance, either cholesterol orsphingomyelin may be added to the liposomal mixture in order to helpstabilize the liposomal structure and to prevent the leakage of theliposomal inner cargo. Further, liposomes are prepared from hydrogenatedegg phosphatidylcholine or egg phosphatidylcholine, cholesterol, anddicetyl phosphate, and their mean vesicle sizes were adjusted to about50 and 100 nm. (see, e.g., Spuch and Navarro, Journal of Drug Delivery,vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679for review).

A liposome formulation may be mainly comprised of natural phospholipidsand lipids such as 1,2-distearoryl-sn-glycero-3-phosphatidyl choline(DSPC), sphingomyelin, egg phosphatidylcholines andmonosialoganglioside. Since this formulation is made up of phospholipidsonly, liposomal formulations have encountered many challenges, one ofthe ones being the instability in plasma. Several attempts to overcomethese challenges have been made, specifically in the manipulation of thelipid membrane. One of these attempts focused on the manipulation ofcholesterol. Addition of cholesterol to conventional formulationsreduces rapid release of the encapsulated bioactive compound into theplasma or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increasesthe stability (see, e.g., Spuch and Navarro, Journal of Drug Delivery,vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679for review).

In a particularly advantageous embodiment, Trojan Horse liposomes (alsoknown as Molecular Trojan Horses) are desirable and protocols may befound at http://cshprotocols.cshlp.org/content/2010/4/pdb.prot5407.long.These particles allow delivery of a transgene to the entire brain afteran intravascular injection. Without being bound by limitation, it isbelieved that neutral lipid particles with specific antibodiesconjugated to surface allow crossing of the blood brain barrier viaendocytosis. Applicant postulates utilizing Trojan Horse Liposomes todeliver the CRISPR family of nucleases to the brain via an intravascularinjection, which would allow whole brain transgenic animals without theneed for embryonic manipulation. About 1-5 g of DNA or RNA may becontemplated for in vivo administration in liposomes.

In another embodiment, the CRISPR Cas system or components thereof maybe administered in liposomes, such as a stable nucleic-acid-lipidparticle (SNALP) (see, e.g., Morrissey et al., Nature Biotechnology,Vol. 23, No. 8, August 2005). Daily intravenous injections of about 1, 3or 5 mg/kg/day of a specific CRISPR Cas targeted in a SNALP arecontemplated. The daily treatment may be over about three days and thenweekly for about five weeks. In another embodiment, a specific CRISPRCas encapsulated SNALP) administered by intravenous injection to atdoses of about 1 or 2.5 mg/kg are also contemplated (see, e.g.,Zimmerman et al., Nature Letters, Vol. 441, 4 May 2006). The SNALPformulation may contain the lipids 3-N-[(wmethoxypoly(ethylene glycol)2000) carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a2:40:10:48 molar percent ratio (see, e.g., Zimmerman et al., NatureLetters, Vol. 441, 4 May 2006).

In another embodiment, stable nucleic-acid-lipid particles (SNALPs) haveproven to be effective delivery molecules to highly vascularizedHepG2-derived liver tumors but not in poorly vascularized HCT-116derived liver tumors (see, e.g., Li, Gene Therapy (2012) 19, 775-780).The SNALP liposomes may be prepared by formulating D-Lin-DMA andPEG-C-DMA with distearoylphosphatidylcholine (DSPC), Cholesterol andsiRNA using a 25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio ofCholesterol/D-Lin-DMA/DSPC/PEG-C-DMA. The resulted SNALP liposomes areabout 80-100 nm in size. In yet another embodiment, a SNALP may comprisesynthetic cholesterol (Sigma-Aldrich, St Louis, Mo., USA),dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, Ala.,USA), 3-N-[(w-methoxy poly(ethyleneglycol)2000)carbamoyl]-1,2-dimyrestyloxypropylamine, and cationic1,2-dilinoleyloxy-3-N,Ndimethylaminopropane (see, e.g., Geisbert et al.,Lancet 2010; 375: 1896-905). A dosage of about 2 mg/kg total CRISPR Casper dose administered as, for example, a bolus intravenous infusion maybe contemplated. In yet another embodiment, a SNALP may comprisesynthetic cholesterol (Sigma-Aldrich),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar LipidsInc.), PEG-cDMA, and 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane(DLinDMA) (see, e.g., Judge, J. Clin. Invest. 119:661-673 (2009)).Formulations used for in vivo studies may comprise a final lipid/RNAmass ratio of about 9:1.

The safety profile of RNAi nanomedicines has been reviewed by Barros andGollob of Alnylam Pharmaceuticals (see, e.g., Advanced Drug DeliveryReviews 64 (2012) 1730-1737). The stable nucleic acid lipid particle(SNALP) is comprised of four different lipids—an ionizable lipid(DLinDMA) that is cationic at low pH, a neutral helper lipid,cholesterol, and a diffusible polyethylene glycol (PEG)-lipid. Theparticle is approximately 80 nm in diameter and is charge-neutral atphysiologic pH. During formulation, the ionizable lipid serves tocondense lipid with the anionic RNA during particle formation. Whenpositively charged under increasingly acidic endosomal conditions, theionizable lipid also mediates the fusion of SNALP with the endosomalmembrane enabling release of RNA into the cytoplasm. The PEG-lipidstabilizes the particle and reduces aggregation during formulation, andsubsequently provides a neutral hydrophilic exterior that improvespharmacokinetic properties.

To date, two clinical programs have been initiated using SNALPformulations with RNA. Tekmira Pharmaceuticals recently completed aphase I single-dose study of SNALP-ApoB in adult volunteers withelevated LDL cholesterol. ApoB is predominantly expressed in the liverand jejunum and is essential for the assembly and secretion of VLDL andLDL. Seventeen subjects received a single dose of SNALP-ApoB (doseescalation across 7 dose levels). There was no evidence of livertoxicity (anticipated as the potential dose-limiting toxicity based onpreclinical studies). One (of two) subjects at the highest doseexperienced flu-like symptoms consistent with immune system stimulation,and the decision was made to conclude the trial.

Alnylam Pharmaceuticals has similarly advanced ALN-TTR01, which employsthe SNALP technology described above and targets hepatocyte productionof both mutant and wild-type TTR to treat TTR amyloidosis (ATTR). ThreeATTR syndromes have been described: familial amyloidotic polyneuropathy(FAP) and familial amyloidotic cardiomyopathy (FAC) both caused byautosomal dominant mutations in TTR; and senile systemic amyloidosis(SSA) cause by wildtype TTR. A placebo-controlled, singledose-escalation phase I trial of ALN-TTR01 was recently completed inpatients with ATTR. ALN-TTR01 was administered as a 15-minute IVinfusion to 31 patients (23 with study drug and 8 with placebo) within adose range of 0.01 to 1.0 mg/kg (based on siRNA). Treatment was welltolerated with no significant increases in liver function tests.Infusion-related reactions were noted in 3 of 23 patients at ≧0.4 mg/kg;all responded to slowing of the infusion rate and all continued onstudy. Minimal and transient elevations of serum cytokines IL-6, IP-10and IL-1ra were noted in two patients at the highest dose of 1 mg/kg (asanticipated from preclinical and NHP studies). Lowering of serum TTR,the expected pharmacodynamics effect of ALN-TTR01, was observed at 1mg/kg.

In yet another embodiment, a SNALP may be made by solubilizing acationic lipid, DSPC, cholesterol and PEG-lipid e.g., in ethanol, e.g.,at a molar ratio of 40:10:40:10, respectively (see, Semple et al.,Nature Niotechnology, Volume 28 Number 2 Feb. 2010, pp. 172-177). Thelipid mixture was added to an aqueous buffer (50 mM citrate, pH 4) withmixing to a final ethanol and lipid concentration of 30% (vol/vol) and6.1 mg/ml, respectively, and allowed to equilibrate at 22° C. for 2 minbefore extrusion. The hydrated lipids were extruded through two stacked80 nm pore-sized filters (Nuclepore) at 22° C. using a Lipex Extruder(Northern Lipids) until a vesicle diameter of 70-90 nm, as determined bydynamic light scattering analysis, was obtained. This generally required1-3 passes. The siRNA (solubilized in a 50 mM citrate, pH 4 aqueoussolution containing 30% ethanol) was added to the pre-equilibrated (35°C.) vesicles at a rate of ˜5 ml/min with mixing. After a final targetsiRNA/lipid ratio of 0.06 (wt/wt) was reached, the mixture was incubatedfor a further 30 min at 35° C. to allow vesicle reorganization andencapsulation of the siRNA. The ethanol was then removed and theexternal buffer replaced with PBS (155 mM NaCl, 3 mM Na₂HPO₄, 1 mMKH₂PO₄, pH 7.5) by either dialysis or tangential flow diafiltration.siRNA were encapsulated in SNALP using a controlled step-wise dilutionmethod process. The lipid constituents of KC2-SNALP were DLin-KC2-DMA(cationic lipid), dipalmitoylphosphatidylcholine (DPPC; Avanti PolarLipids), synthetic cholesterol (Sigma) and PEG-C-DMA used at a molarratio of 57.1:7.1:34.3:1.4. Upon formation of the loaded particles,SNALP were dialyzed against PBS and filter sterilized through a 0.2 μmfilter before use. Mean particle sizes were 75-85 nm and 90-95% of thesiRNA was encapsulated within the lipid particles. The final siRNA/lipidratio in formulations used for in vivo testing was ˜0.15 (wt/wt).LNP-siRNA systems containing Factor VII siRNA were diluted to theappropriate concentrations in sterile PBS immediately before use and theformulations were administered intravenously through the lateral tailvein in a total volume of 10 ml/kg. This method and these deliverysystems may be extrapolated to the CRISPR Cas system of the presentinvention.

Other Lipids

Other cationic lipids, such as amino lipid2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) maybe utilized to encapsulate CRISPR Cas system or components thereof ornucleic acid molecule(s) coding therefor e.g., similar to SiRNA (see,e.g., Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529-8533), and hencemay be employed in the practice of the invention. A preformed vesiclewith the following lipid composition may be contemplated: amino lipid,di stearoylphosphatidylcholine (DSPC), cholesterol and(R)-2,3-bis(octadecyloxy) propyl-1-(methoxy poly(ethyleneglycol)2000)propylcarbamate (PEG-lipid) in the molar ratio 40/10/40/10,respectively, and a FVII siRNA/total lipid ratio of approximately 0.05(w/w). To ensure a narrow particle size distribution in the range of70-90 nm and a low polydispersity index of 0.11±0.04 (n=56), theparticles may be extruded up to three times through 80 nm membranesprior to adding the CRISPR Cas RNA. Particles containing the highlypotent amino lipid 16 may be used, in which the molar ratio of the fourlipid components 16, DSPC, cholesterol and PEG-lipid (50/10/38.5/1.5)which may be further optimized to enhance in vivo activity.

Michael S D Kormann et al. (“Expression of therapeutic proteins afterdelivery of chemically modified mRNA in mice: Nature Biotechnology,Volume: 29, Pages: 154-157 (2011)) describes the use of lipid envelopesto deliver RNA. Use of lipid envelopes is also preferred in the presentinvention.

In another embodiment, lipids may be formulated with the CRISPR Cassystem of the present invention to form lipid nanoparticles (LNPs).Lipids include, but are not limited to, DLin-KC2-DMA4, C12-200 andcolipids disteroylphosphatidyl choline, cholesterol, and PEG-DMG may beformulated with CRISPR Cas instead of siRNA (see, e.g., Novobrantseva,Molecular Therapy—Nucleic Acids (2012) 1, e4; doi:10.1038/mtna.2011.3)using a spontaneous vesicle formation procedure. The component molarratio may be about 50/10/38.5/1.5 (DLin-KC2-DMA orC12-200/disteroylphosphatidyl choline/cholesterol/PEG-DMG). The finallipid:siRNA weight ratio may be ˜12:1 and 9:1 in the case ofDLin-KC2-DMA and C12-200 lipid nanoparticles (LNPs), respectively. Theformulations may have mean particle diameters of ˜80 nm with >90%entrapment efficiency. A 3 mg/kg dose may be contemplated.

Tekmira has a portfolio of approximately 95 patent families, in the U.S.and abroad, that are directed to various aspects of LNPs and LNPformulations (see, e.g., U.S. Pat. Nos. 7,982,027; 7,799,565; 8,058,069;8,283,333; 7,901,708; 7,745,651; 7,803,397; 8,101,741; 8,188,263;7,915,399; 8,236,943 and 7,838,658 and European Pat. Nos 1766035;1519714; 1781593 and 1664316), all of which may be used and/or adaptedto the present invention.

The CRISPR Cas system or components thereof or nucleic acid molecule(s)coding therefor may be delivered encapsulated in PLGA Microspheres suchas that further described in US published applications 20130252281 and20130245107 and 20130244279 (assigned to Moderna Therapeutics) whichrelate to aspects of formulation of compositions comprising modifiednucleic acid molecules which may encode a protein, a protein precursor,or a partially or fully processed form of the protein or a proteinprecursor. The formulation may have a molar ratio 50:10:38.5:1.5-3.0(cationic lipid:fusogenic lipid:cholesterol:PEG lipid). The PEG lipidmay be selected from, but is not limited to PEG-c-DOMG, PEG-DMG. Thefusogenic lipid may be DSPC. See also, Schrum et al., Delivery andFormulation of Engineered Nucleic Acids, US published application20120251618.

Nanomerics' technology addresses bioavailability challenges for a broadrange of therapeutics, including low molecular weight hydrophobic drugs,peptides, and nucleic acid based therapeutics (plasmid, siRNA, miRNA).Specific administration routes for which the technology has demonstratedclear advantages include the oral route, transport across theblood-brain-barrier, delivery to solid tumours, as well as to the eye.See, e.g., Mazza et al., 2013, ACS Nano. 2013 Feb. 26; 7(2):1016-26;Uchegbu and Siew, 2013, J Pharm Sci. 102(2):305-10 and Lalatsa et al.,2012, J Control Release. 2012 Jul. 20; 161(2):523-36.

U.S. Patent Publication No. 20050019923 describes cationic dendrimersfor delivering bioactive molecules, such as polynucleotide molecules,peptides and polypeptides and/or pharmaceutical agents, to a mammalianbody. The dendrimers are suitable for targeting the delivery of thebioactive molecules to, for example, the liver, spleen, lung, kidney orheart (or even the brain). Dendrimers are synthetic 3-dimensionalmacromolecules that are prepared in a step-wise fashion from simplebranched monomer units, the nature and functionality of which can beeasily controlled and varied. Dendrimers are synthesised from therepeated addition of building blocks to a multifunctional core(divergent approach to synthesis), or towards a multifunctional core(convergent approach to synthesis) and each addition of a 3-dimensionalshell of building blocks leads to the formation of a higher generationof the dendrimers. Polypropylenimine dendrimers start from adiaminobutane core to which is added twice the number of amino groups bya double Michael addition of acrylonitrile to the primary aminesfollowed by the hydrogenation of the nitriles. This results in adoubling of the amino groups. Polypropylenimine dendrimers contain 100%protonable nitrogens and up to 64 terminal amino groups (generation 5,DAB 64). Protonable groups are usually amine groups which are able toaccept protons at neutral pH. The use of dendrimers as gene deliveryagents has largely focused on the use of the polyamidoamine. andphosphorous containing compounds with a mixture of amine/amide orN—P(O₂)S as the conjugating units respectively with no work beingreported on the use of the lower generation polypropylenimine dendrimersfor gene delivery. Polypropylenimine dendrimers have also been studiedas pH sensitive controlled release systems for drug delivery and fortheir encapsulation of guest molecules when chemically modified byperipheral amino acid groups. The cytotoxicity and interaction ofpolypropylenimine dendrimers with DNA as well as the transfectionefficacy of DAB 64 has also been studied.

U.S. Patent Publication No. 20050019923 is based upon the observationthat, contrary to earlier reports, cationic dendrimers, such aspolypropylenimine dendrimers, display suitable properties, such asspecific targeting and low toxicity, for use in the targeted delivery ofbioactive molecules, such as genetic material. In addition, derivativesof the cationic dendrimer also display suitable properties for thetargeted delivery of bioactive molecules. See also, Bioactive Polymers,US published application 20080267903, which discloses “Various polymers,including cationic polyamine polymers and dendrimeric polymers, areshown to possess anti-proliferative activity, and may therefore beuseful for treatment of disorders characterised by undesirable cellularproliferation such as neoplasms and tumours, inflammatory disorders(including autoimmune disorders), psoriasis and atherosclerosis. Thepolymers may be used alone as active agents, or as delivery vehicles forother therapeutic agents, such as drug molecules or nucleic acids forgene therapy. In such cases, the polymers' own intrinsic anti-tumouractivity may complement the activity of the agent to be delivered.” Thedisclosures of these patent publications may be employed in conjunctionwith herein teachings for delivery of CRISPR Cas system(s) orcomponent(s) thereof or nucleic acid molecule(s) coding therefor.

Supercharged Proteins

Supercharged proteins are a class of engineered or naturally occurringproteins with unusually high positive or negative net theoretical chargeand may be employed in delivery of CRISPR Cas system(s) or component(s)thereof or nucleic acid molecule(s) coding therefor. Bothsupernegatively and superpositively charged proteins exhibit aremarkable ability to withstand thermally or chemically inducedaggregation. Superpositively charged proteins are also able to penetratemammalian cells. Associating cargo with these proteins, such as plasmidDNA, RNA, or other proteins, can enable the functional delivery of thesemacromolecules into mammalian cells both in vitro and in vivo. DavidLiu's lab reported the creation and characterization of superchargedproteins in 2007 (Lawrence et al., 2007, Journal of the AmericanChemical Society 129, 10110-10112).

The nonviral delivery of RNA and plasmid DNA into mammalian cells arevaluable both for research and therapeutic applications (Akinc et al.,2010, Nat. Biotech. 26, 561-569). Purified +36 GFP protein (or othersuperpositively charged protein) is mixed with RNAs in the appropriateserum-free media and allowed to complex prior addition to cells.Inclusion of serum at this stage inhibits formation of the superchargedprotein-RNA complexes and reduces the effectiveness of the treatment.The following protocol has been found to be effective for a variety ofcell lines (McNaughton et al., 2009, Proc. Natl. Acad. Sci. USA 106,6111-6116). However, pilot experiments varying the dose of protein andRNA should be performed to optimize the procedure for specific celllines.

(1) One day before treatment, plate 1×10⁵ cells per well in a 48-wellplate.

(2) On the day of treatment, dilute purified +36 GFP protein inserumfree media to a final concentration 200 nM. Add RNA to a finalconcentration of 50 nM. Vortex to mix and incubate at room temperaturefor 10 min.

(3) During incubation, aspirate media from cells and wash once with PBS.

(4) Following incubation of +36 GFP and RNA, add the protein-RNAcomplexes to cells.

(5) Incubate cells with complexes at 37° C. for 4 h.

(6) Following incubation, aspirate the media and wash three times with20 U/mL heparin PBS. Incubate cells with serum-containing media for afurther 48 h or longer depending upon the assay for activity.

(7) Analyze cells by immunoblot, qPCR, phenotypic assay, or otherappropriate method.

David Liu's lab has further found +36 GFP to be an effective plasmiddelivery reagent in a range of cells. As plasmid DNA is a larger cargothan siRNA, proportionately more +36 GFP protein is required toeffectively complex plasmids. For effective plasmid delivery Applicantshave developed a variant of +36 GFP bearing a C-terminal HA2 peptidetag, a known endosome-disrupting peptide derived from the influenzavirus hemagglutinin protein. The following protocol has been effectivein a variety of cells, but as above it is advised that plasmid DNA andsupercharged protein doses be optimized for specific cell lines anddelivery applications.

(1) One day before treatment, plate 1×10⁵ per well in a 48-well plate.

(2) On the day of treatment, dilute purified

36 GFP protein in serumfree media to a final concentration 2 mM. Add 1mg of plasmid DNA. Vortex to mix and incubate at room temperature for 10min.

(3) During incubation, aspirate media from cells and wash once with PBS.

(4) Following incubation of

36 GFP and plasmid DNA, gently add the protein-DNA complexes to cells.

(5) Incubate cells with complexes at 37 C for 4 h.

(6) Following incubation, aspirate the media and wash with PBS. Incubatecells in serum-containing media and incubate for a further 24-48 h.

(7) Analyze plasmid delivery (e.g., by plasmid-driven gene expression)as appropriate.

David Liu's lab has further found +36 GFP to be an effective plasmiddelivery reagent in a range of cells. As plasmid DNA is a larger cargothan siRNA, proportionately more +36 GFP protein is required toeffectively complex plasmids. For effective plasmid delivery Applicantshave developed a variant of +36 GFP bearing a C-terminal HA2 peptidetag, a known endosome-disrupting peptide derived from the influenzavirus hemagglutinin protein. The following protocol has been effectivein a variety of cells, but as above it is advised that plasmid DNA andsupercharged protein doses be optimized for specific cell lines anddelivery applications: (1) One day before treatment, plate 1×10⁵ perwell in a 48-well plate. (2) On the day of treatment, dilute purified

36 GFP protein in serumfree media to a final concentration 2 mM. Add 1mg of plasmid DNA. Vortex to mix and incubate at room temperature for 10min. (3) During incubation, aspirate media from cells and wash once withPBS. (4) Following incubation of

36 GFP and plasmid DNA, gently add the protein-DNA complexes to cells.(5) Incubate cells with complexes at 37 C for 4 h. (6) Followingincubation, aspirate the media and wash with PBS. Incubate cells inserum-containing media and incubate for a further 24-48 h. (7) Analyzeplasmid delivery (e.g., by plasmid-driven gene expression) asappropriate. See also, e.g., McNaughton et al., Proc. Natl. Acad. Sci.USA 106, 6111-6116 (2009); Cronican et al., ACS Chemical Biology 5,747-752 (2010); Cronican et al., Chemistry & Biology 18, 833-838 (2011);Thompson et al., Methods in Enzymology 503, 293-319 (2012); Thompson, D.B., et al., Chemistry & Biology 19 (7), 831-843 (2012). The methods ofthe super charged proteins may be used and/or adapted for delivery ofthe CRISPR Cas system of the present invention. These systems of Dr. Luiand documents herein in inconjunction with herein teachints can beemployed in the delivery of CRISPR Cas system(s) or component(s) thereofor nucleic acid molecule(s) coding therefor.

Cell Penetrating Peptides (CPPs)

In yet another embodiment, cell penetrating peptides (CPPs) arecontemplated for the delivery of the CRISPR Cas system. CPPs are shortpeptides that facilitate cellular uptake of various molecular cargo(from nanosize particles to small chemical molecules and large fragmentsof DNA). The term “cargo” as used herein includes but is not limited tothe group consisting of therapeutic agents, diagnostic probes, peptides,nucleic acids, antisense oligonucleotides, plasmids, proteins,nanoparticles, liposomes, chromophores, small molecules and radioactivematerials. In aspects of the invention, the cargo may also comprise anycomponent of the CRISPR Cas system or the entire functional CRISPR Cassystem. Aspects of the present invention further provide methods fordelivering a desired cargo into a subject comprising: (a) preparing acomplex comprising the cell penetrating peptide of the present inventionand a desired cargo, and (b) orally, intraarticularly,intraperitoneally, intrathecally, intrarterially, intranasally,intraparenchymally, subcutaneously, intramuscularly, intravenously,dermally, intrarectally, or topically administering the complex to asubject. The cargo is associated with the peptides either throughchemical linkage via covalent bonds or through non-covalentinteractions.

The function of the CPPs are to deliver the cargo into cells, a processthat commonly occurs through endocytosis with the cargo delivered to theendosomes of living mammalian cells. Cell-penetrating peptides are ofdifferent sizes, amino acid sequences, and charges but all CPPs have onedistinct characteristic, which is the ability to translocate the plasmamembrane and facilitate the delivery of various molecular cargoes to thecytoplasm or an organelle. CPP translocation may be classified intothree main entry mechanisms: direct penetration in the membrane,endocytosis-mediated entry, and translocation through the formation of atransitory structure. CPPs have found numerous applications in medicineas drug delivery agents in the treatment of different diseases includingcancer and virus inhibitors, as well as contrast agents for celllabeling. Examples of the latter include acting as a carrier for GFP, MMcontrast agents, or quantum dots. CPPs hold great potential as in vitroand in vivo delivery vectors for use in research and medicine. CPPstypically have an amino acid composition that either contains a highrelative abundance of positively charged amino acids such as lysine orarginine or has sequences that contain an alternating pattern ofpolar/charged amino acids and non-polar, hydrophobic amino acids. Thesetwo types of structures are referred to as polycationic or amphipathic,respectively. A third class of CPPs are the hydrophobic peptides,containing only a polar residues, with low net charge or havehydrophobic amino acid groups that are crucial for cellular uptake. Oneof the initial CPPs discovered was the trans-activating transcriptionalactivator (Tat) from Human Immunodeficiency Virus 1 (HIV-1) which wasfound to be efficiently taken up from the surrounding media by numerouscell types in culture. Since then, the number of known CPPs has expandedconsiderably and small molecule synthetic analogues with more effectiveprotein transduction properties have been generated. CPPs include butare not limited to Penetratin, Tat (48-60), Transportan, and (R-AhX-R4)(Ahx=aminohexanoyl).

U.S. Pat. No. 8,372,951, provides a CPP derived from eosinophil cationicprotein (ECP) which exhibits highly cell-penetrating efficiency and lowtoxicity. Aspects of delivering the CPP with its cargo into a vertebratesubject are also provided. Further aspects of CPPs and their deliveryare described in U.S. Pat. Nos. 8,575,305; 8,614,194 and 8,044,019. CPPscan be used to deliver the CRISPR-Cas system or components thereof. ThatCPPs can be employed to deliver the CRISPR-Cas system or componentsthereof is also provided in the manuscript “Gene disruption bycell-penetrating peptide-mediated delivery of Cas9 protein and guideRNA”, by Suresh Ramakrishna, Abu-Bonsrah Kwaku Dad, Jagadish Beloor, etal. Genome Res. 2014 Apr. 2. [Epub ahead of print], incorporated byreference in its entirety, wherein it is demonstrated that treatmentwith CPP-conjugated recombinant Cas9 protein and CPP-complexed guideRNAs lead to endogenous gene disruptions in human cell lines. In thepaper the Cas9 protein was conjugated to CPP via a thioether bond,whereas the guide RNA was complexed with CPP, forming condensed,positively charged nanoparticles. It was shown that simultaneous andsequential treatment of human cells, including embryonic stem cells,dermal fibroblasts, HEK293T cells, HeLa cells, and embryonic carcinomacells, with the modified Cas9 and guide RNA led to efficient genedisruptions with reduced off-target mutations relative to plasmidtransfections.

Implantable Devices

In another embodiment, implantable devices are also contemplated fordelivery of the CRISPR Cas system or component(s) thereof or nucleicacid molecule(s) coding therefor. For example, U.S. Patent Publication20110195123 discloses an implantable medical device which elutes a druglocally and in prolonged period is provided, including several types ofsuch a device, the treatment modes of implementation and methods ofimplantation. The device comprising of polymeric substrate, such as amatrix for example, that is used as the device body, and drugs, and insome cases additional scaffolding materials, such as metals oradditional polymers, and materials to enhance visibility and imaging. Animplantable delivery device can be advantageous in providing releaselocally and over a prolonged period, where drug is released directly tothe extracellular matrix (ECM) of the diseased area such as tumor,inflammation, degeneration or for symptomatic objectives, or to injuredsmooth muscle cells, or for prevention. One kind of drug is RNA, asdisclosed above, and this system may be used/and or adapted to theCRISPR Cas system of the present invention. The modes of implantation insome embodiments are existing implantation procedures that are developedand used today for other treatments, including brachytherapy and needlebiopsy. In such cases the dimensions of the new implant described inthis invention are similar to the original implant. Typically a fewdevices are implanted during the same treatment procedure.

As in U.S. Patent Publication 20110195123, there is provided a drugdelivery implantable or insertable system, including systems applicableto a cavity such as the abdominal cavity and/or any other type ofadministration in which the drug delivery system is not anchored orattached, comprising a biostable and/or degradable and/or bioabsorbablepolymeric substrate, which may for example optionally be a matrix. Itshould be noted that the term “insertion” also includes implantation.The drug delivery system is preferably implemented as a “Loder” asdescribed in U.S. Patent Publication 20110195123.

The polymer or plurality of polymers are biocompatible, incorporating anagent and/or plurality of agents, enabling the release of agent at acontrolled rate, wherein the total volume of the polymeric substrate,such as a matrix for example, in some embodiments is optionally andpreferably no greater than a maximum volume that permits a therapeuticlevel of the agent to be reached. As a non-limiting example, such avolume is preferably within the range of 0.1 m³ to 1000 mm³, as requiredby the volume for the agent load. The Loder may optionally be larger,for example when incorporated with a device whose size is determined byfunctionality, for example and without limitation, a knee joint, anintra-uterine or cervical ring and the like.

The drug delivery system (for delivering the composition) is designed insome embodiments to preferably employ degradable polymers, wherein themain release mechanism is bulk erosion; or in some embodiments, nondegradable, or slowly degraded polymers are used, wherein the mainrelease mechanism is diffusion rather than bulk erosion, so that theouter part functions as membrane, and its internal part functions as adrug reservoir, which practically is not affected by the surroundingsfor an extended period (for example from about a week to about a fewmonths). Combinations of different polymers with different releasemechanisms may also optionally be used. The concentration gradient atthe surface is preferably maintained effectively constant during asignificant period of the total drug releasing period, and therefore thediffusion rate is effectively constant (termed “zero mode” diffusion).By the term “constant” it is meant a diffusion rate that is preferablymaintained above the lower threshold of therapeutic effectiveness, butwhich may still optionally feature an initial burst and/or mayfluctuate, for example increasing and decreasing to a certain degree.The diffusion rate is preferably so maintained for a prolonged period,and it can be considered constant to a certain level to optimize thetherapeutically effective period, for example the effective silencingperiod.

The drug delivery system optionally and preferably is designed to shieldthe nucleotide based therapeutic agent from degradation, whetherchemical in nature or due to attack from enzymes and other factors inthe body of the subject.

The drug delivery system of U.S. Patent Publication 20110195123 isoptionally associated with sensing and/or activation appliances that areoperated at and/or after implantation of the device, by non and/orminimally invasive methods of activation and/oracceleration/deceleration, for example optionally including but notlimited to thermal heating and cooling, laser beams, and ultrasonic,including focused ultrasound and/or RF (radiofrequency) methods ordevices.

According to some embodiments of U.S. Patent Publication 20110195123,the site for local delivery may optionally include target sitescharacterized by high abnormal proliferation of cells, and suppressedapoptosis, including tumors, active and or chronic inflammation andinfection including autoimmune diseases states, degenerating tissueincluding muscle and nervous tissue, chronic pain, degenerative sites,and location of bone fractures and other wound locations for enhancementof regeneration of tissue, and injured cardiac, smooth and striatedmuscle.

The site for implantation of the composition, or target site, preferablyfeatures a radius, area and/or volume that is sufficiently small fortargeted local delivery. For example, the target site optionally has adiameter in a range of from about 0.1 mm to about 5 cm.

The location of the target site is preferably selected for maximumtherapeutic efficacy. For example, the composition of the drug deliverysystem (optionally with a device for implantation as described above) isoptionally and preferably implanted within or in the proximity of atumor environment, or the blood supply associated thereof.

For example the composition (optionally with the device) is optionallyimplanted within or in the proximity to pancreas, prostate, breast,liver, via the nipple, within the vascular system and so forth.

The target location is optionally selected from the group consisting of(as non-limiting examples only, as optionally any site within the bodymay be suitable for implanting a Loder): 1. brain at degenerative siteslike in Parkinson or Alzheimer disease at the basal ganglia, white andgray matter; 2. spine as in the case of amyotrophic lateral sclerosis(ALS); 3. uterine cervix to prevent HPV infection; 4. active and chronicinflammatory joints; 5. dermis as in the case of psoriasis; 6.sympathetic and sensoric nervous sites for analgesic effect; 7. Intraosseous implantation; 8. acute and chronic infection sites; 9. Intravaginal; 10. Inner ear—auditory system, labyrinth of the inner ear,vestibular system; 11. Intra tracheal; 12. Intra-cardiac; coronary,epicardiac; 13. urinary bladder; 14. biliary system; 15. parenchymaltissue including and not limited to the kidney, liver, spleen; 16. lymphnodes; 17. salivary glands; 18. dental gums; 19. Intra-articular (intojoints); 20. Intra-ocular; 21. Brain tissue; 22. Brain ventricles; 23.Cavities, including abdominal cavity (for example but withoutlimitation, for ovary cancer); 24. Intra esophageal and 25. Intrarectal.

Optionally insertion of the system (for example a device containing thecomposition) is associated with injection of material to the ECM at thetarget site and the vicinity of that site to affect local pH and/ortemperature and/or other biological factors affecting the diffusion ofthe drug and/or drug kinetics in the ECM, of the target site and thevicinity of such a site.

Optionally, according to some embodiments, the release of said agentcould be associated with sensing and/or activation appliances that areoperated prior and/or at and/or after insertion, by non and/or minimallyinvasive and/or else methods of activation and/oracceleration/deceleration, including laser beam, radiation, thermalheating and cooling, and ultrasonic, including focused ultrasound and/orRF (radiofrequency) methods or devices, and chemical activators.

According to other embodiments of U.S. Patent Publication 20110195123,the drug preferably comprises a RNA, for example for localized cancercases in breast, pancreas, brain, kidney, bladder, lung, and prostate asdescribed below. Although exemplified with RNAi, many drugs areapplicable to be encapsulated in Loder, and can be used in associationwith this invention, as long as such drugs can be encapsulated with theLoder substrate, such as a matrix for example, and this system may beused and/or adapted to deliver the CRISPR Cas system of the presentinvention.

As another example of a specific application, neuro and musculardegenerative diseases develop due to abnormal gene expression. Localdelivery of RNAs may have therapeutic properties for interfering withsuch abnormal gene expression. Local delivery of anti apoptotic, antiinflammatory and anti degenerative drugs including small drugs andmacromolecules may also optionally be therapeutic. In such cases theLoder is applied for prolonged release at constant rate and/or through adedicated device that is implanted separately. All of this may be usedand/or adapted to the CRISPR Cas system of the present invention.

As yet another example of a specific application, psychiatric andcognitive disorders are treated with gene modifiers. Gene knockdown is atreatment option. Loders locally delivering agents to central nervoussystem sites are therapeutic options for psychiatric and cognitivedisorders including but not limited to psychosis, bi-polar diseases,neurotic disorders and behavioral maladies. The Loders could alsodeliver locally drugs including small drugs and macromolecules uponimplantation at specific brain sites. All of this may be used and/oradapted to the CRISPR Cas system of the present invention.

As another example of a specific application, silencing of innate and/oradaptive immune mediators at local sites enables the prevention of organtransplant rejection. Local delivery of RNAs and immunomodulatingreagents with the Loder implanted into the transplanted organ and/or theimplanted site renders local immune suppression by repelling immunecells such as CD8 activated against the transplanted organ. All of thismay be used/and or adapted to the CRISPR Cas system of the presentinvention.

As another example of a specific application, vascular growth factorsincluding VEGFs and angiogenin and others are essential forneovascularization. Local delivery of the factors, peptides,peptidomimetics, or suppressing their repressors is an importanttherapeutic modality; silencing the repressors and local delivery of thefactors, peptides, macromolecules and small drugs stimulatingangiogenesis with the Loder is therapeutic for peripheral, systemic andcardiac vascular disease.

The method of insertion, such as implantation, may optionally already beused for other types of tissue implantation and/or for insertions and/orfor sampling tissues, optionally without modifications, or alternativelyoptionally only with non-major modifications in such methods. Suchmethods optionally include but are not limited to brachytherapy methods,biopsy, endoscopy with and/or without ultrasound, such as ERCP,stereotactic methods into the brain tissue, Laparoscopy, includingimplantation with a laparoscope into joints, abdominal organs, thebladder wall and body cavities.

Implantable device technology herein discussed can be employed withherein teachings and hence by this disclosure and the knowledge in theart, CRISPR-Cas system or components thereof or nucleic acid moleculesthereof or encoding or providing components may be delivered via animplantable device.

Patient-Specific Screening Methods

A CRISPR-Cas system that targets nucleotide, e.g., trinucleotide repeatscan be used to screen patients or patent samples for the presence ofsuch repeats. The repeats can be the target of the RNA of the CRISPR-Cassystem, and if there is binding thereto by the CRISPR-Cas system, thatbinding can be detected, to thereby indicate that such a repeat ispresent. Thus, a CRISPR-Cas system can be used to screen patients orpatient samples for the presence of the repeat. The patient can then beadministered suitable compound(s) to address the condition; or, can beadministed a CRISPR-Cas system to bind to and cause insertion, deletionor mutation and alleviate the condition.

HSC—Delivery to and Editing of Hematopoetic Stem Cells; and ParticularConditions

The term “Hematopoetic Stem Cell” or “HSC” is meant to include broadlythose cells considered to be an HSC, e.g., blood cells that give rise toall the other blood cells and are derived from mesoderm; located in thered bone marrow, which is contained in the core of most bones. HSCs ofthe invention include cells having a phenotype of hematopoeitic stemcells, identified by small size, lack of lineage (lin) markers, andmarkers that belong to the cluster of differentiation series, like:CD34, CD38, CD90, CD133, CD105, CD45, and also c-kit, —the receptor forstem cell factor. Hematopoietic stem cells are negative for the markersthat are used for detection of lineage commitment, and are, thus, calledLin−; and, during their purification by FACS, a number of up to 14different mature blood-lineage markers, e.g., CD13 & CD33 for myeloid,CD71 for erythroid, CD19 for B cells, CD61 for megakaryocytic, etc. forhumans; and, B220 (murine CD45) for B cells, Mac-1 (CD11b/CD18) formonocytes, Gr-1 for Granulocytes, Ter119 for erythroid cells, Il7Ra,CD3, CD4, CD5, CD8 for T cells, etc. Mouse HSC markers: CD34^(lo/−),SCA-1+, Thy1.1^(+/lo), CD38+, C-kit+, lin−, and Human HSC markers:CD34+, CD59+, Thy1/CD90+, CD38lo/−, C-kit/CD117+, and lin−. HSCs areidentified by markers. Hence in embodiments discussed herein, the HSCscan be CD34+ cells. HSCs can also be hematopoietic stem cells that areCD34−/CD38−. Stem cells that may lack c-kit on the cell surface that areconsidered in the art as HSCs are within the ambit of the invention, aswell as CD133+ cells likewise considered HSCs in the art.

The CRISPR-Cas9 system has been engineered to target genetic locus orloci in HSCs. Cas9 protein, advantageously codon-optimized for aeukaryotic cell and especially a mammalian cell, e.g., a human cell, forinstance, HSC, and sgRNA targeting a locus or loci in HSC, e.g., thegene EMX1, were prepared. These were advantageously delivered viaparticles. The particles were formed by the Cas9 protein and the sgRNAbeing admixed. The sgRNA and Cas9 protein mixture was admixed with amixture comprising or consisting essentially of or consisting ofsurfactant, phospholipid, biodegradable polymer, lipoprotein andalcohol, whereby particles containing the sgRNA and Cas9 protein wereformed. The invention comprehends so making particles and particles fromsuch a method as well as uses thereof.

More generally, particles were formed using an efficient process. First,Cas9 protein and sgRNA targeting the gene EMX1 or the control gene LacZwere mixed together at a suitable, e.g., 3:1 to 1:3 or 2:1 to 1:2 or 1:1molar ratio, at a suitable temperature, e.g., 15-30 C, e.g., 20-25 C,e.g., room temperature, for a suitable time, e.g., 15-45, such as 30minutes, advantageously in sterile, nuclease free buffer, e.g., 1×PBS.Separately, particle components such as or comprising: a surfactant,e.g., cationic lipid, e.g., 1,2-dioleoyl-3-trimethylammonium-propane(DOTAP); phospholipid, e.g., dimyristoylphosphatidylcholine (DMPC);biodegradable polymer, such as an ethylene-glycol polymer or PEG, and alipoprotein, such as a low-density lipoprotein, e.g., cholesterol weredissolved in an alcohol, advantageously a C₁₋₆ alkyl alcohol, such asmethanol, ethanol, isopropanol, e.g., 100% ethanol. The two solutionswere mixed together to form particles containing the Cas9-sgRNAcomplexes. In certain embodiments the particle can contain an HDRtemplate. That can be a particle co-administered with sgRNA+Cas9protein-containing particle, or i.e., in addition to contacting an HSCwith an sgRNA+Cas9 protein-containing particle, the HSC is contactedwith a particle containing an HDR template; or the HSC is contacted witha particle containing all of the sgRNA, Cas9 and the HDR template. TheHDR template can be administered by a separate vector, whereby in afirst instance the particle penetrates an HSC cell and the separatevector also penetrates the cell, wherein the HSC genome is modified bythe sgRNA+Cas9 and the HDR template is also present, whereby a genomicloci is modified by the HDR; for instance, this may result in correctinga mutation.

After the particles were formed, HSCs in 96 well plates were transfectedwith 15 ug Cas9 protein per well. Three days after transfection, HSCswere harvested, and the number of insertions and deletions (indels) atthe EMX1 locus were quantified.

This demonstrates that HSCs can be modified using CRISPR-Cas9 targetinga genomic locus or loci of interest in the HSC. The HSCs that are to bemodified can be in vivo, i.e., in an organism, for example a human or anon-human eukaryote, e.g., animal, such as fish, e.g., zebra fish,mammal, e.g., primate, e.g., ape, chimpanzee, macaque, rodent, e.g.,mouse, rabbit, rat, canine or dog, livestock (cow/bovine, sheep/ovine,goat or pig), fowl or poultry, e.g., chicken. The HSCs that are to bemodified can be in vitro, i.e., outside of such an organism. And,modified HSCs can be used ex vivo, i.e., one or more HSCs of such anorganism can be obtained or isolated from the organism, optionally theHSC(s) can be expanded, the HSC(s) are modified by a compositioncomprising a CRISPR-Cas9 that targets a genetic locus or loci in theHSC, e.g., by contacting the HSC(s) with the composition, for instance,wherein the composition comprises a particle containing the CRISPRenzyme and one or more sgRNA that targets the genetic locus or loci inthe HSC, such as a particle obtained or obtainable from admixing ansgRNA and Cas9 protein mixture with a mixture comprising or consistingessentially of or consisting of surfactant, phospholipid, biodegradablepolymer, lipoprotein and alcohol (wherein one or more sgRNA targets thegenetic locus or loci in the HSC), optionally expanding the resultantmodified HSCs and administering to the organism the resultant modifiedHSCs. In some instances the isolated or obtained HSCs can be from afirst organism, such as an organism from a same species as a secondorganism, and the second organism can be the organism to which the theresultant modified HSCs are administered, e.g., the first organism canbe a donor (such as a relative as in a parent or sibling) to the secondorganism. Modified HSCs can have genetic modifications to address oralleviate or reduce symptoms of a disease or condition state of anindividual or subject or patient. Modified HSCs, e.g., in the instanceof a first organism donor to a second organism, can have geneticmodifications to have the HSCs have one or more proteins e.g. surfacemarkers or proteins more like that of the second organism. Modified HSCscan have genetic modifications to simulate a a disease or conditionstate of an individual or subject or patient and would bere-administered to a non-human organism so as to prepare an animalmodel. Expansion of HSCs is within the ambit of the skilled person fromthis disclosure and knowledge in the art, see e.g., Lee, “Improved exvivo expansion of adult hematopoietic stem cells by overcomingCUL4-mediated degradation of HOXB4.” Blood. 2013 May 16; 121(20):4082-9.doi: 10.1182/blood-2012-09-455204. Epub 2013 Mar. 21.

As indicated to improve activity, sgRNA may be pre-complexed with theCas9 protein, before formulating the entire complex in a particle.Formulations may be made with a different molar ratio of differentcomponents known to promote delivery of nucleic acids into cells (e.g.1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC), polyethyleneglycol (PEG), and cholesterol) For example DOTAP:DMPC:PEG:CholesterolMolar Ratios may be DOTAP 100, DMPC 0, PEG 0, Cholesterol 0; or DOTAP90, DMPC 0, PEG 10, Cholesterol 0; or DOTAP 90, DMPC 0, PEG 5,Cholesterol 5. DOTAP 100, DMPC 0, PEG 0, Cholesterol 0. The inventionaccordingly comprehends admixing sgRNA, Cas9 protein and components thatform a particle; as well as particles from such admixing.

In a preferred embodiment, particles containing the Cas9-sgRNA complexesmay be formed by mixing Cas9 protein and one or more sgRNAs together,preferably at a 1:1 molar ratio, enzyme: guide RNA. Separately, thedifferent components known to promote delivery of nucleic acids (e.g.DOTAP, DMPC, PEG, and cholesterol) are dissolved, preferably in ethanol.The two solutions are mixed together to form particles containing theCas9-sgRNA complexes. After the particles are formed, Cas9-sgRNAcomplexes may be transfected into cells (e.g. HSCs). Bar coding may beapplied. The particles, the Cas-9 and/or the sgRNA may be barcoded.

The invention in an embodiment comprehends a method of preparing ansgRNA-and-Cas9 protein containing particle comprising admixing an sgRNAand Cas9 protein mixture with a mixture comprising or consistingessentially of or consisting of surfactant, phospholipid, biodegradablepolymer, lipoprotein and alcohol. An embodiment comprehends ansgRNA-and-Cas9 protein containing particle from the method. Theinvention in an embodiment comprehends use of the particle in a methodof modifying a genomic locus of interest, or an organism or a non-humanorganism by manipulation of a target sequence in a genomic locus ofinterest, comprising contacting a cell containing the genomic locus ofinterest with the particle wherein the sgRNA targets the genomic locusof interest; or a method of modifying a genomic locus of interest, or anorganism or a non-human organism by manipulation of a target sequence ina genomic locus of interest, comprising contacting a cell containing thegenomic locus of interest with the particle wherein the sgRNA targetsthe genomic locus of interest. In these embodiments, the genomic locusof interest is advantageously a genomic locus in an HSC.

Considerations for Therapeutic Applications:

A consideration in genome editing therapy is the choice ofsequence-specific nuclease. Each nuclease platform possesses its ownunique set of strengths and weaknesses, many of which must be balancedin the context of treatment to maximize therapeutic benefit (FIG. 1).Thus far, two therapeutic editing approaches with nucleases have shownsignificant promise: gene disruption and gene correction. Genedisruption involves stimulation of NHEJ to create targeted indels ingenetic elements, often resulting in loss of function mutations that arebeneficial to patients (FIG. 2A). In contrast, gene correction uses HDRto directly reverse a disease causing mutation, restoring function whilepreserving physiological regulation of the corrected element (FIG. 2B).HDR may also be used to insert a therapeutic transgene into a defined‘safe harbor’ locus in the genome to recover missing gene function (FIG.2C). For a specific editing therapy to be efficacious, a sufficientlyhigh level of modification must be achieved in target cell populationsto reverse disease symptoms. This therapeutic modification ‘threshold’is determined by the fitness of edited cells following treatment and theamount of gene product necessary to reverse symptoms. With regard tofitness, editing creates three potential outcomes for treated cellsrelative to their unedited counterparts: increased, neutral, ordecreased fitness. In the case of increased fitness, for example in thetreatment of SCID-X1, modified hematopoietic progenitor cellsselectively expand relative to their unedited counterparts. SCID-X1 is adisease caused by mutations in the IL2RG gene, the function of which isrequired for proper development of the hematopoietic lymphocyte lineage[Leonard, W. J., et al. Immunological reviews 138, 61-86 (1994);Kaushansky, K. & Williams, W. J. Williams hematology, (McGraw-HillMedical, New York, 2010)]. In clinical trials with patients who receivedviral gene therapy for SCID-X1, and a rare example of a spontaneouscorrection of SCID-X1 mutation, corrected hematopoietic progenitor cellswere able to overcome this developmental block and expand relative totheir diseased counterparts to mediate therapy [Bousso, P., et al.Proceedings of the National Academy of Sciences of the United States ofAmerica 97, 274-278 (2000); Hacein-Bey-Abina, S., et al. The New Englandjournal of medicine 346, 1185-1193 (2002); Gaspar, H. B., et al. Lancet364, 2181-2187 (2004)]. In this case, where edited cells possess aselective advantage, even low numbers of edited cells can be amplifiedthrough expansion, providing a therapeutic benefit to the patient. Incontrast, editing for other hematopoietic diseases, like chronicgranulomatous disorder (CGD), would induce no change in fitness foredited hematopoietic progenitor cells, increasing the therapeuticmodification threshold. CGD is caused by mutations in genes encodingphagocytic oxidase proteins, which are normally used by neutrophils togenerate reactive oxygen species that kill pathogens [Mukherjee, S. &Thrasher, A. J. Gene 525, 174-181 (2013)]. As dysfunction of these genesdoes not influence hematopoietic progenitor cell fitness or development,but only the ability of a mature hematopoietic cell type to fightinfections, there would be likely no preferential expansion of editedcells in this disease. Indeed, no selective advantage for gene correctedcells in CGD has been observed in gene therapy trials, leading todifficulties with long-term cell engraftment [Malech, H. L., et al.Proceedings of the National Academy of Sciences of the United States ofAmerica 94, 12133-12138 (1997); Kang, H. J., et al. Molecular therapy:the journal of the American Society of Gene Therapy 19, 2092-2101(2011)]. As such, significantly higher levels of editing would berequired to treat diseases like CGD, where editing creates a neutralfitness advantage, relative to diseases where editing creates increasedfitness for target cells. If editing imposes a fitness disadvantage, aswould be the case for restoring function to a tumor suppressor gene incancer cells, modified cells would be outcompeted by their diseasedcounterparts, causing the benefit of treatment to be low relative toediting rates. This latter class of diseases would be particularlydifficult to treat with genome editing therapy.

In addition to cell fitness, the amount of gene product necessary totreat disease also influences the minimal level of therapeutic genomeediting that must be achieved to reverse symptoms. Haemophilia B is onedisease where a small change in gene product levels can result insignificant changes in clinical outcomes. This disease is caused bymutations in the gene encoding factor IX, a protein normally secreted bythe liver into the blood, where it functions as a component of theclotting cascade. Clinical severity of haemophilia B is related to theamount of factor IX activity. Whereas severe disease is associated withless than 1% of normal activity, milder forms of the diseases areassociated with greater than 1% of factor IX activity [Kaushansky, K. &Williams, W. J. Williams hematology, (McGraw-Hill Medical, New York,2010); Lofqvist, T., et al. Journal of internal medicine 241, 395-400(1997)]. This suggests that editing therapies that can restore factor IXexpression to even a small percentage of liver cells could have a largeimpact on clinical outcomes. A study using ZFNs to correct a mouse modelof haemophilia B shortly after birth demonstrated that 3-7% correctionwas sufficient to reverse disease symptoms, providing preclinicalevidence for this hypothesis [Li, H., et al. Nature 475, 217-221(2011)].

Disorders where a small change in gene product levels can influenceclinical outcomes and diseases where there is a fitness advantage foredited cells, are ideal targets for genome editing therapy, as thetherapeutic modification threshold is low enough to permit a high chanceof success given the current technology. Targeting these diseases hasnow resulted in successes with editing therapy at the preclinical leveland a phase I clinical trial. Improvements in DSB repair pathwaymanipulation and nuclease delivery are needed to extend these promisingresults to diseases with a neutral fitness advantage for edited cells,or where larger amounts of gene product are needed for treatment. TheTable below shows some examples of applications of genome editing totherapeutic models, and the references of the below Table and thedocuments cited in those references are hereby incorporated herein byreference as if set out in full.

Nuclease Platform Therapeutic Disease Type Employed Strategy ReferencesHemophilia B ZFN HDR-mediated Li, H., et al. insertion of correct Nature475, gene sequence 217-221 (2011) SCID ZFN HDR-mediated Genovese, P., etal. insertion of correct Nature 510, gene sequence 235-240 (2014)Hereditary CRISPR HDR-mediated Yin, H., et al. tyrosinemia correction ofmutation Nature in liver biotechnology 32, 551-553 (2014)

Addressing each of the conditions of the foreging table, using theCRISPR-Cas9 system to target by either HDR-mediated correction ofmutation, or HDR-mediated insertion of correct gene sequence,advantageously via a delivery system as herein, e.g., a particledelivery system, is within the ambit of the skilled person from thisdisclosure and the knowledge in the art. Thus, an embodiment comprehendscontacting a Hemophilia B, SCID (e.g., SCID-X1, ADA-SCID) or Hereditarytyrosinemia mutation-carrying HSC with an sgRNA-and-Cas9 proteincontaining particle targeting a genomic locus of interest as toHemophilia B, SCID (e.g., SCID-X1, ADA-SCID) or Hereditary tyrosinemia(e.g., as in Li, Genovese or Yin). The particle also can contain asuitable HDR template to correct the mutation; or the HSC can becontacted with a second particle or a vector that contains or deliversthe HDR template. In this regard, it is mentioned that Haemophilia B isan X-linked recessive disorder caused by loss-of-function mutations inthe gene encoding Factor IX, a crucial component of the clottingcascade. Recovering Factor IX activity to above 1% of its levels inseverely affected individuals can transform the disease into asignificantly milder form, as infusion of recombinant Factor IX intosuch patients prophylactically from a young age to achieve such levelslargely ameliorates clinical complications. With the knowledge in theart and the teachings in this disclosure, the skilled person can correctHSCs as to Haemophilia B using a CRISPR-Cas9 system that targets andcorrects the mutation (X-linked recessive disorder caused byloss-of-function mutations in the gene encoding Factor IX) (e.g., with asuitable HDR template that delivers a coding sequence for Factor IX);specifically, the sgRNA can target mutation that give rise toHaemophilia B, and the HDR can provide coding for proper expression ofFactor IX. An sgRNA that targets the mutation-and-Cas9 proteincontaining particle is contacted with HSCs carrying the mutation. Theparticle also can contain a suitable HDR template to correct themutation for proper expression of Factor IX; or the HSC can be contactedwith a second particle or a vector that contains or delivers the HDRtemplate. The so contacted cells can be administered; and optionallytreated/expanded; cf. Cartier, discussed herein.

Severe Combined Immune Deficiency (SCID) results from a defect inlymphocytes T maturation, always associated with a functional defect inlymphocytes B (Cavazzana-Calvo et al., Annu. Rev. Med., 2005, 56,585-602; Fischer et al., Immunol. Rev., 2005, 203, 98-109). Overallincidence is estimated to 1 in 75 000 births. Patients with untreatedSCID are subject to multiple opportunist micro-organism infections, anddo generally not live beyond one year. SCID can be treated by allogenichematopoietic stem cell transfer, from a familial donor.Histocompatibility with the donor can vary widely. In the case ofAdenosine Deaminase (ADA) deficiency, one of the SCID forms, patientscan be treated by injection of recombinant Adenosine Deaminase enzyme.Since the ADA gene has been shown to be mutated in SCID patients(Giblett et al., Lancet, 1972, 2, 1067-1069), several other genesinvolved in SCID have been identified (Cavazzana-Calvo et al., Annu.Rev. Med., 2005, 56, 585-602; Fischer et al., Immunol. Rev., 2005, 203,98-109). There are four major causes for SCID: (i) the most frequentform of SCID, SCID-X1 (X-linked SCID or X-SCID), is caused by mutationin the IL2RG gene, resulting in the absence of mature T lymphocytes andNK cells. IL2RG encodes the gamma C protein (Noguchi, et al., Cell,1993, 73, 147-157), a common component of at least five interleukinreceptor complexes. These receptors activate several targets through theJAK3 kinase (Macchi et al., Nature, 1995, 377, 65-68), whichinactivation results in the same syndrome as gamma C inactivation; (ii)mutation in the ADA gene results in a defect in purine metabolism thatis lethal for lymphocyte precursors, which in turn results in the quasiabsence of B, T and NK cells; (iii) V(D)J recombination is an essentialstep in the maturation of immunoglobulins and T lymphocytes receptors(TCRs). Mutations in Recombination Activating Gene 1 and 2 (RAG1 andRAG2) and Artemis, three genes involved in this process, result in theabsence of mature T and B lymphocytes; and (iv) Mutations in other genessuch as CD45, involved in T cell specific signaling have also beenreported, although they represent a minority of cases (Cavazzana-Calvoet al., Annu. Rev. Med., 2005, 56, 585-602; Fischer et al., Immunol.Rev., 2005, 203, 98-109). Since when their genetic bases have beenidentified, the different SCID forms have become a paradigm for genetherapy approaches (Fischer et al., Immunol. Rev., 2005, 203, 98-109)for two major reasons. First, as in all blood diseases, an ex vivotreatment can be envisioned. Hematopoietic Stem Cells (HSCs) can berecovered from bone marrow, and keep their pluripotent properties for afew cell divisions. Therefore, they can be treated in vitro, and thenreinjected into the patient, where they repopulate the bone marrow.Second, since the maturation of lymphocytes is impaired in SCIDpatients, corrected cells have a selective advantage. Therefore, a smallnumber of corrected cells can restore a functional immune system. Thishypothesis was validated several times by (i) the partial restoration ofimmune functions associated with the reversion of mutations in SCIDpatients (Hirschhorn et al., Nat. Genet., 1996, 13, 290-295; Stephan etal., N. Engl. J. Med., 1996, 335, 1563-1567; Bousso et al., Proc. Natl.,Acad. Sci. USA, 2000, 97, 274-278; Wada et al., Proc. Natl. Acad. Sci.USA, 2001, 98, 8697-8702; Nishikomori et al., Blood, 2004, 103,4565-4572), (ii) the correction of SCID-X1 deficiencies in vitro inhematopoietic cells (Candotti et al., Blood, 1996, 87, 3097-3102;Cavazzana-Calvo et al., Blood, 1996, Blood, 88, 3901-3909; Taylor etal., Blood, 1996, 87, 3103-3107; Hacein-Bey et al., Blood, 1998, 92,4090-4097), (iii) the correction of SCID-X1 (Soudais et al., Blood,2000, 95, 3071-3077; Tsai et al., Blood, 2002, 100, 72-79), JAK-3(Bunting et al., Nat. Med., 1998, 4, 58-64; Bunting et al., Hum. GeneTher., 2000, 11, 2353-2364) and RAG2 (Yates et al., Blood, 2002, 100,3942-3949) deficiencies in vivo in animal models and (iv) by the resultof gene therapy clinical trials (Cavazzana-Calvo et al., Science, 2000,288, 669-672; Aiuti et al., Nat. Med., 2002; 8, 423-425; Gaspar et al.,Lancet, 2004, 364, 2181-2187). U.S. Patent Publication No. 20110182867assigned to the Children's Medical Center Corporation and the Presidentand Fellows of Harvard College relates to methods and uses of modulatingfetal hemoglobin expression (HbF) in a hematopoietic progenitor cellsvia inhibitors of BCL11A expression or activity, such as RNAi andantibodies. The targets disclosed in U.S. Patent Publication No.20110182867, such as BCL11A, may be targeted by the CRISPR Cas system ofthe present invention for modulating fetal hemoglobin expression. Seealso Bauer et al. (Science 11 Oct. 2013: Vol. 342 no. 6155 pp. 253-257)and Xu et al. (Science 18 Nov. 2011: Vol. 334 no. 6058 pp. 993-996) foradditional BCL11A targets. Using a CRISPR-Cas9 system that targets andone or more of the mutations associated with SCID, for instance aCRISPR-Cas9 system having sgRNA(s) and HDR template(s) that respectivelytargets mutation of IL2RG that give rise to SCID and provide correctiveexpression of the gamma C protein. An sgRNA that targets the mutation(s)(e.g., one or more involved in SCID, for instance mutation as to IL2RGthat encodes the gamma C protein)-and-Cas9 protein containing particleis contacted with HSCs carrying the mutation(s). The particle also cancontain a suitable HDR template(s) to correct the mutation for properexpression of one or more of the proteins involved in SCID, e.g., gammaC protein; or the HSC can be contacted with a second particle or avector that contains or delivers the HDR template. The so contactedcells can be administered; and optionally treated/expanded; cf. Cartier,discussed herein. Mention is also made of target sequences identified inUS Patent Publication Nos. 20110225664, 20110091441, 20100229252,20090271881 and 20090222937 that may be of interest as to the presentinvention.

In Cartier, “MINI-SYMPOSIUM: X-Linked Adrenoleukodystrophypa,Hematopoietic Stem Cell Transplantation and Hematopoietic Stem Cell GeneTherapy in X-Linked Adrenoleukodystrophy,” Brain Pathology 20 (2010)857-862, incorporated herein by reference along with the documents itcites, as if set out in full, there is recognition that allogeneichematopoietic stem cell transplantation (HSCT) was utilized to delivernormal lysosomal enzyme to the brain of a patient with Hurler's disease,and a discussion of HSC gene therapy to treat ALD. In two patients,peripheral CD34+cells were collected after granulocyte-colonystimulating factor (G-CSF) mobilization and transduced with anmyeloproliferative sarcoma virus enhancer, negative control regiondeleted, d1587rev primer binding site substituted (MND)-ALD lentiviralvector. CD34+ cells from the patients were transduced with the MND-ALDvector during 16 h in the presence of cytokines at low concentrations.Transduced CD34+ cells were frozen after transduction to perform on 5%of cells various safety tests that included in particular threereplication-competent lentivirus (RCL) assays. Transduction efficacy ofCD34+ cells ranged from 35% to 50% with a mean number of lentiviralintegrated copy between 0.65 and 0.70. After the thawing of transducedCD34+ cells, the patients were reinfused with more than 4.10⁶ transducedCD34+ cells/kg following full myeloablation with busulfan andcyclophos-phamide. The patient's HSCs were ablated to favor engraftmentof the gene-corrected HSCs. Hematological recovery occurred between days13 and 15 for the two patients. Nearly complete immunological recoveryoccurred at 12 months for the first patient, and at 9 months for thesecond patient. In contrast to using lentivirus, with the knowledge inthe art and the teachings in this disclosure, the skilled person cancorrect HSCs as to ALD using a CRISPR-Cas9 system that targets andcorrects the mutation (e.g., with a suitable HDR template);specifically, the sgRNA can target mutations in ABCD1, a gene located onthe X chromosome that codes for ALD, a peroxisomal membrane transporterprotein, and the HDR can provide coding for proper expression of theprotein. An sgRNA that targets the mutation-and-Cas9 protein containingparticle is contacted with HSCs, e.g., CD34+ cells carrying the mutationas in Cartier. The particle also can contain a suitable HDR template tocorrect the mutation for expression of the peroxisomal membranetransporter protein; or the HSC can be contacted with a second particleor a vector that contains or delivers the HDR template. The so contactedcells optionally can be treated as in Cartier. The so contacted cellscan be administered as in Cartier.

Drakopoulou, “Review Article, The Ongoing Challenge of HematopoieticStem Cell-Based Gene Therapy for β-Thalassemia,” Stem CellsInternational, Volume 2011, Article ID 987980, 10 pages,doi:10.4061/2011/987980, incorporated herein by reference along with thedocuments it cites, as if set out in full, discuss modifying HSCs usinga lentivirus that delivers a gene for β-globin or γ-globin. In contrastto using lentivirus, with the knowledge in the art and the teachings inthis disclosure, the skilled person can correct HSCs as to β-Thalassemiausing a CRISPR-Cas9 system that targets and corrects the mutation (e.g.,with a suitable HDR template that delivers a coding sequence forβ-globin or γ-globin, advantageously non-sickling β-globin or γ-globin);specifically, the sgRNA can target mutation that give rise toβ-Thalassemia, and the HDR can provide coding for proper expression ofβ-globin or γ-globin. An sgRNA that targets the mutation-and-Cas9protein containing particle is contacted with HSCs carrying themutation. The particle also can contain a suitable HDR template tocorrect the mutation for proper expression of β-globin or γ-globin; orthe HSC can be contacted with a second particle or a vector thatcontains or delivers the HDR template. The so contacted cells can beadministered; and optionally treated/expanded; cf. Cartier. In thisregard mention is made of: Cavazzana, “Outcomes of Gene Therapy forβ-Thalassemia Major via Transplantation of Autologous Hematopoietic StemCells Transduced Ex Vivo with a Lentiviral β^(A-T87Q)-Globin Vector.”tif2014. org/abstractFiles/Jean%20Antoine%20Ribeil_Abstract.pdf;Cavazzana-Calvo, “Transfusion independence and HMGA2 activation aftergene therapy of human β-thalassaemia”, Nature 467, 318-322 (16 Sep.2010) doi:10.1038/nature09328; Nienhuis, “Development of Gene Therapyfor Thalassemia, Cold Spring Harbor Perpsectives in Medicine, doi:10.1101/cshperspect.a011833 (2012), LentiGlobin BB305, a lentiviralvector containing an engineered β-globin gene (βA-T87Q); and Xie et al.,“Seamless gene correction of β-thalassaemia mutations inpatient-specific iPSCs using CRISPR/Cas9 and piggyback” Genome Researchgr.173427.114 (2014) http://www.genome.org/cgi/doi/10.1101/gr.173427.114(Cold Spring Harbor Laboratory Press); that is the subject of Cavazzanawork involving human β-thalassaemia and the subject of the Xie work, areall incorporated herein by reference, together with all documents citedtherein or associated therewith. In the instant invention, the HDRtemplate can provide for the HSC to express an engineered β-globin gene(e.g., βA-T87Q), or β-globin as in Xie.

Song et al. (Stem Cells Dev. 2015 May 1; 24(9):1053-65. doi:10.1089/scd.2014.0347. Epub 2015 Feb. 5) used CRISPR/Cas9 to correctβ-Thal iPSCs; gene-corrected cells exhibit normal karyotypes and fullpluripotency as human embryonic stem cells (hESCs) showed nooff-targeting effects. Then, Song et al. evaluated the differentiationefficiency of the gene-corrected β-Thal iPSCs. Song et al. found thatduring hematopoietic differentiation, gene-corrected β-Thal iPSCs showedan increased embryoid body ratio and various hematopoietic progenitorcell percentages. More importantly, the gene-corrected β-Thal iPSC linesrestored HBB expression and reduced reactive oxygen species productioncompared with the uncorrected group. Song et al.'s study suggested thathematopoietic differentiation efficiency of β-Thal iPSCs was greatlyimproved once corrected by the CRISPR-Cas9 system. Similar methods maybe performed utilizing the CRISPR-Cas systems described herein, e.g.systems comprising Cas9 effector proteins.

Mention is made of WO 2015/148860, through the teachings herein theinvention comprehends methods and materials of these documents appliedin conjunction with the teachings herein. In an aspect of blood-relateddisease gene therapy, methods and compositions for treating betathalassemia may be adapted to the CRISPR-Cas system of the presentinvention (see, e.g., WO 2015/148860). In an embodiment, WO 2015/148860involves the treatment or prevention of beta thalassemia, or itssymptoms, e.g., by altering the gene for B-cell CLL/lymphoma 11A(BCL11A). The BCL11A gene is also known as B-cell CLL/lymphoma 11A,BCL11A-L, BCL11A-S, BCL11AXL, CTIP 1, HBFQTL5 and ZNF. BCL11A encodes azinc-finger protein that is involved in the regulation of globin geneexpression. By altering the BCL11A gene (e.g., one or both alleles ofthe BCL11A gene), the levels of gamma globin can be increased. Gammaglobin can replace beta globin in the hemoglobin complex and effectivelycarry oxygen to tissues, thereby ameliorating beta thalassemia diseasephenotypes.

Sickle cell anemia is an autosomal recessive genetic disease in whichred blood cells become sickle-shaped. It is caused by a single basesubstitution in the β-globin gene, which is located on the short arm ofchromosome 11. As a result, valine is produced instead of glutamic acidcausing the production of sickle hemoglobin (HbS). This results in theformation of a distorted shape of the erythrocytes. Due to this abnormalshape, small blood vessels can be blocked, causing serious damage to thebone, spleen and skin tissues. This may lead to episodes of pain,frequent infections, hand-foot syndrome or even multiple organ failure.The distorted erythrocytes are also more susceptible to hemolysis, whichleads to serious anemia. As in the case of β-thalassaemia, sickle cellanemia can be corrected by modifying HSCs with the CRISPR/Cas9 system.The system allows the specific editing of the cell's genome by cuttingits DNA and then letting it repair itself. The Cas9 protein is insertedand directed by a RNA guide to the mutated point and then it cuts theDNA at that point. Simultaneously, a healthy version of the sequence isinserted. This sequence is used by the cell's own repair system to fixthe induced cut. In this way, the CRISPR/Cas9 allows the correction ofthe mutation in the previously obtained stem cells. With the knowledgein the art and the teachings in this disclosure, the skilled person cancorrect HSCs as to sickle cell anemia using a CRISPR-Cas9 system thattargets and corrects the mutation (e.g., with a suitable HDR templatethat delivers a coding sequence for β-globin, advantageouslynon-sickling β-globin); specifically, the sgRNA can target mutation thatgive rise to sickle cell anemia, and the HDR can provide coding forproper expression of β-globin. An sgRNA that targets themutation-and-Cas9 protein containing particle is contacted with HSCscarrying the mutation. The particle also can contain a suitable HDRtemplate to correct the mutation for proper expression of β-globin; orthe HSC can be contacted with a second particle or a vector thatcontains or delivers the HDR template. The so contacted cells can beadministered; and optionally treated/expanded; cf. Cartier. The HDRtemplate can provide for the HSC to express an engineered β-globin gene(e.g., βA-T87Q), or β-globin as in Xie.

Mention is also made of WO 2015/148863 and through the teachings hereinthe invention comprehends methods and materials of these documents whichmay be adapted to the CRISPR-Cas system of the present invention. In anaspect of treating and preventing sickle cell disease, which is aninherited hematologic disease, WO 2015/148863 comprehends altering theBCL11A gene. By altering the BCL11A gene (e.g., one or both alleles ofthe BCL11A gene), the levels of gamma globin can be increased. Gammaglobin can replace beta globin in the hemoglobin complex and effectivelycarry oxygen to tissues, thereby ameliorating sickle cell diseasephenotypes.

Williams, “Broadening the Indications for Hematopoietic Stem CellGenetic Therapies,” Cell Stem Cell 13:263-264 (2013), incorporatedherein by reference along with the documents it cites, as if set out infull, report lentivirus-mediated gene transfer into HSC/P cells frompatients with the lysosomal storage disease metachromatic leukodystrophydisease (MLD), a genetic disease caused by deficiency of arylsulfatase A(ARSA), resulting in nerve demyelination; and lentivirus-mediated genetransfer into HSCs of patients with Wiskott-Aldrich syndrome (WAS)(patients with defective WAS protein, an effector of the small GTPaseCDC42 that regulates cytoskeletal function in blood cell lineages andthus suffer from immune deficiency with recurrent infections, autoimmunesymptoms, and thrombocytopenia with abnormally small and dysfunctionalplatelets leading to excessive bleeding and an increased risk ofleukemia and lymphoma). In contrast to using lentivirus, with theknowledge in the art and the teachings in this disclosure, the skilledperson can correct HSCs as to MLD (deficiency of arylsulfatase A (ARSA))using a CRISPR-Cas9 system that targets and corrects the mutation(deficiency of arylsulfatase A (ARSA)) (e.g., with a suitable HDRtemplate that delivers a coding sequence for ARSA); specifically, thesgRNA can target mutation that gives rise to MLD (deficient ARSA), andthe HDR can provide coding for proper expression of ARSA. An sgRNA thattargets the mutation-and-Cas9 protein containing particle is contactedwith HSCs carrying the mutation. The particle also can contain asuitable HDR template to correct the mutation for proper expression ofARSA; or the HSC can be contacted with a second particle or a vectorthat contains or delivers the HDR template. The so contacted cells canbe administered; and optionally treated/expanded; cf. Cartier. Incontrast to using lentivirus, with the knowledge in the art and theteachings in this disclosure, the skilled person can correct HSCs as toWAS using a CRISPR-Cas9 system that targets and corrects the mutation(deficiency of WAS protein) (e.g., with a suitable HDR template thatdelivers a coding sequence for WAS protein); specifically, the sgRNA cantarget mutation that gives rise to WAS (deficient WAS protein), and theHDR can provide coding for proper expression of WAS protein. An sgRNAthat targets the mutation-and-Cas9 protein containing particle iscontacted with HSCs carrying the mutation. The particle also can containa suitable HDR template to correct the mutation for proper expression ofWAS protein; or the HSC can be contacted with a second particle or avector that contains or delivers the HDR template. The so contactedcells can be administered; and optionally treated/expanded; cf. Cartier.

In an aspect of the invention, methods and compositions which involveediting a target nucleic acid sequence, or modulating expression of atarget nucleic acid sequence, and applications thereof in connectionwith cancer immunotherapy are comprehended by adapting the CRISPR-Cassystem of the present invention. Reference is made to the application ofgene therapy in WO 2015/161276 which involves methods and compositionswhich can be used to affect T-cell proliferation, survival and/orfunction by altering one or more T-cell expressed genes, e.g., one ormore of FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, TRAC and/or TRBC genes. Ina related aspect, T-cell proliferation can be affected by altering oneor more T-cell expressed genes, e.g., the CBLB and/or PTPN6 gene, FASand/or BID gene, CTLA4 and/or PDCDI and/or TRAC and/or TRBC gene.

Chimeric antigen receptor (CAR)19 T-cells exhibit anti-leukemic effectsin patient malignancies. However, leukemia patients often do not haveenough T-cells to collect, meaning that treatment must involve modifiedT cells from donors. Accordingly, there is interest in establishing abank of donor T-cells. Qasim et al. (“First Clinical Application ofTalen Engineered Universal CAR19 T Cells in B-ALL” ASH 57th AnnualMeeting and Exposition, Dec. 5-8, 2015, Abstract 2046(https://ash.confex.com/ash/2015/web program/Paper81653. html publishedonline November 2015) discusses modifying CAR19 T cells to eliminate therisk of graft-versus-host disease through the disruption of T-cellreceptor expression and CD52 targeting. Furthermore, CD52 cells weretargeted such that they became insensitive to Alemtuzumab, and thusallowed Alemtuzumab to prevent host-mediated rejection of humanleukocyte antigen (HLA) mismatched CAR19 T-cells. Investigators usedthird generation self-inactivating lentiviral vector encoding a 4g7CAR19 (CD19 scFv-4-1BB-CD3ζ) linked to RQR8, then electroporated cellswith two pairs of TALEN mRNA for multiplex targeting for both the T-cellreceptor (TCR) alpha constant chain locus and the CD52 gene locus. Cellswhich were still expressing TCR following ex vivo expansion weredepleted using CliniMacs α/β TCR depletion, yielding a T-cell product(UCART19) with <1% TCR expression, 85% of which expressed CAR19, and 64%becoming CD52 negative. The modified CAR19 T cells were administered totreat a patient's relapsed acute lymphoblastic leukemia. The teachingsprovided herein provide effective methods for providing modifiedhematopoietic stem cells and progeny thereof, including but not limitedto cells of the myeloid and lymphoid lineages of blood, including Tcells, B cells, monocytes, macrophages, neutrophils, basophils,eosinophils, erythrocytes, dendritic cells, and megakaryocytes orplatelets, and natural killer cells and their precursors andprogenitors. Such cells can be modified by knocking out, knocking in, orotherwise modulating targets, for example to remove or modulate CD52 asdescribed above, and other targets, such as, without limitation, CXCR4,and PD-1. Thus compositions, cells, and method of the invention can beused to can be to modulate immune responses and to treat, withoutlimitation, malignancies, viral infections, and immune disorders, inconjunction with modification of administration of T cells or othercells to patients.

Watts, “Hematopoietic Stem Cell Expansion and Gene Therapy” Cytotherapy13(10):1164-1171. doi:10.3109/14653249.2011.620748 (2011), incorporatedherein by reference along with the documents it cites, as if set out infull, discusses hematopoietic stem cell (HSC) gene therapy, e.g.,virus-mediated HSC gene therapy, as an highly attractive treatmentoption for many disorders including hematologic conditions,immunodeficiencies including HIV/AIDS, and other genetic disorders likelysosomal storage diseases, including SCID-X1, ADA-SCID, β-thalassemia,X-linked CGD, Wiskott-Aldrich syndrome, Fanconi anemia,adrenoleukodystrophy (ALD), and metachromatic leukodystrophy (MLD).

With the knowledge in the art and the teachings in this disclosure, theskilled person can correct HSCs as to a genetic hematologic disorder,e.g., β-Thalassemia, Hemophilia, or a genetic lysosomal storage disease.

With the knowledge in the art and the teachings in this disclosure theskilled person can correct HSCs as to immunodeficiency condition such asHIV/AIDS comprising contacting an HSC with a CRISPR-Cas9 system thattargets and knocks out CCR5. An sgRNA (and advantageously a dual guideapproach, e.g., a pair of different sgRNAs; for instance, sgRNAstargeting of two clinically relevant genes, B2M and CCR5, in primaryhuman CD4+ T cells and CD34+ hematopoietic stem and progenitor cells(HSPCs)) that targets and knocks out CCR5-and-Cas9 protein containingparticle is contacted with HSCs. The so contacted cells can beadministered; and optionally treated/expanded; cf. Cartier. See alsoKiem, “Hematopoietic stem cell-based gene therapy for HIV disease,” CellStem Cell. Feb. 3, 2012; 10(2): 137-147; incorporated herein byreference along with the documents it cites; Mandal et al, “EfficientAblation of Genes in Human Hematopoietic Stem and Effector Cells usingCRISPR/Cas9,” Cell Stem Cell, Volume 15, Issue 5, p643-652, 6 Nov. 2014;incorporated herein by reference along with the documents it cites.Mention is also made of Ebina, “CRISPR/Cas9 system to suppress HIV-1expression by editing HIV-1 integrated proviral DNA” SCIENTIFIC REPORTS3: 2510 DOI: 10.1038/srep02510, incorporated herein by reference alongwith the documents it cites, as another means for combating HIV/AIDSusing a CRISPR-Cas9 system.

The rationale for genome editing for HIV treatment originates from theobservation that individuals homozygous for loss of function mutationsin CCR5, a cellular co-receptor for the virus, are highly resistant toinfection and otherwise healthy, suggesting that mimicking this mutationwith genome editing could be a safe and effective therapeutic strategy[Liu, R., et al. Cell 86, 367-377 (1996)]. This idea was clinicallyvalidated when an HIV infected patient was given an allogeneic bonemarrow transplant from a donor homozygous for a loss of function CCR5mutation, resulting in undetectable levels of HIV and restoration ofnormal CD4 T-cell counts [Hutter, G., et al. The New England journal ofmedicine 360, 692-698 (2009)]. Although bone marrow transplantation isnot a realistic treatment strategy for most HIV patients, due to costand potential graft vs. host disease, HIV therapies that convert apatient's own T-cells into CCR5 are desirable.

Early studies using ZFNs and NHEJ to knockout CCR5 in humanized mousemodels of HIV showed that transplantation of CCR5 edited CD4 T cellsimproved viral load and CD4 T-cell counts [Perez, E. E., et al. Naturebiotechnology 26, 808-816 (2008)]. Importantly, these models also showedthat HIV infection resulted in selection for CCR5 null cells, suggestingthat editing confers a fitness advantage and potentially allowing asmall number of edited cells to create a therapeutic effect.

As a result of this and other promising preclinical studies, genomeediting therapy that knocks out CCR5 in patient T cells has now beentested in humans [Holt, N., et al. Nature biotechnology 28, 839-847(2010); Li, L., et al. Molecular therapy: the journal of the AmericanSociety of Gene Therapy 21, 1259-1269 (2013)]. In a recent phase Iclinical trial, CD4+ T cells from patients with HIV were removed, editedwith ZFNs designed to knockout the CCR5 gene, and autologouslytransplanted back into patients [Tebas, P., et al. The New Englandjournal of medicine 370, 901-910 (2014)].

In another study (Mandal et al., Cell Stem Cell, Volume 15, Issue 5,p643-652, 6 Nov. 2014), CRISPR-Cas9 has targeted two clinical relevantgenes, B2M and CCR5, in human CD4+ T cells and CD34+ hematopoietic stemand progenitor cells (HSPCs). Use of single RNA guides led to highlyefficient mutagenesis in HSPCs but not in T cells. A dual guide approachimproved gene deletion efficacy in both cell types. HSPCs that hadundergone genome editing with CRISPR-Cas9 retained multilineagepotential. Predicted on- and off-target mutations were examined viatarget capture sequencing in HSPCs and low levels of off-targetmutagenesis were observed at only one site. These results demonstratethat CRISPR-Cas9 can efficiently ablate genes in HSPCs with minimaloff-target mutagenesis, which have broad applicability for hematopoieticcell-based therapy.

Wang et al. (PLoS One. 2014 Dec. 26; 9(12):e115987. doi:10.1371/journal.pone.0115987) silenced CCR5 via CRISPR associatedprotein 9 (Cas9) and single guided RNAs (guide RNAs) with lentiviralvectors expressing Cas9 and CCR5 guide RNAs. Wang et al. showed that asingle round transduction of lentiviral vectors expressing Cas9 and CCR5guide RNAs into HIV-1 susceptible human CD4+ cells yields highfrequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are notonly resistant to R5-tropic HIV-1, including transmitted/founder (T/F)HIV-1 isolates, but also have selective advantage over CCR5gene-undisrupted cells during R5-tropic HIV-1 infection. Genomemutations at potential off-target sites that are highly homologous tothese CCR5 guide RNAs in stably transduced cells even at 84 days posttransduction were not detected by a T7 endonuclease I assay.

Fine et al. (Sci Rep. 2015 Jul. 1; 5:10777. doi: 10.1038/srep10777)identified a two-cassette system expressing pieces of the S. pyogenesCas9 (SpCas9) protein which splice together in cellula to form afunctional protein capable of site-specific DNA cleavage. With specificCRISPR guide strands, Fine et al. demonstrated the efficacy of thissystem in cleaving the HBB and CCR5 genes in human HEK-293T cells as asingle Cas9 and as a pair of Cas9 nickases. The trans-spliced SpCas9(tsSpCas9) displayed ˜35% of the nuclease activity compared with thewild-type SpCas9 (wtSpCas9) at standard transfection doses, but hadsubstantially decreased activity at lower dosing levels. The greatlyreduced open reading frame length of the tsSpCas9 relative to wtSpCas9potentially allows for more complex and longer genetic elements to bepackaged into an AAV vector including tissue-specific promoters,multiplexed guide RNA expression, and effector domain fusions to SpCas9.

Li et al. (J Gen Virol. 2015 August; 96(8):2381-93. doi:10.1099/vir.0.000139. Epub 2015 Apr. 8) demonstrated that CRISPR-Cas9can efficiently mediate the editing of the CCR5 locus in cell lines,resulting in the knockout of CCR5 expression on the cell surface.Next-generation sequencing revealed that various mutations wereintroduced around the predicted cleavage site of CCR5. For each of thethree most effective guide RNAs that were analyzed, no significantoff-target effects were detected at the 15 top-scoring potential sites.By constructing chimeric Ad5F35 adenoviruses carrying CRISPR-Cas9components, Li et al. efficiently transduced primary CD4+ T-lymphocytesand disrupted CCR5 expression, and the positively transduced cells wereconferred with HIV-1 resistance.

Mention is made of WO 2015/148670 and through the teachings herein theinvention comprehends methods and materials of this document applied inconjunction with the teachings herein. In an aspect of gene therapy,methods and compositions for editing of a target sequence related to orin connection with Human Immunodeficiency Virus (HIV) and AcquiredImmunodeficiency Syndrome (AIDS) are comprehended. In a related aspect,the invention described herein comprehends prevention and treatment ofHIV infection and AIDS, by introducing one or more mutations in the genefor C-C chemokine receptor type 5 (CCR5). The CCR5 gene is also known asCKR5, CCR-5, CD195, CKR-5, CCCKR5, CMKBR5, IDDM22, and CC-CKR-5. In afurther aspect, the invention described herein comprehends provide forprevention or reduction of HIV infection and/or prevention or reductionof the ability for HIV to enter host cells, e.g., in subjects who arealready infected. Exemplary host cells for HIV include, but are notlimited to, CD4 cells, T cells, gut associated lymphatic tissue (GALT),macrophages, dendritic cells, myeloid precursor cell, and microglia.Viral entry into the host cells requires interaction of the viralglycoproteins gp41 and gp120 with both the CD4 receptor and aco-receptor, e.g., CCR5. If a co-receptor, e.g., CCR5, is not present onthe surface of the host cells, the virus cannot bind and enter the hostcells. The progress of the disease is thus impeded. By knocking out orknocking down CCR5 in the host cells, e.g., by introducing a protectivemutation (such as a CCR5 delta 32 mutation), entry of the HIV virus intothe host cells is prevented.

One of skill in the art may utilize the above studies of, for example,Holt, N., et al. Nature biotechnology 28, 839-847 (2010), Li, L., et al.Molecular therapy: the journal of the American Society of Gene Therapy21, 1259-1269 (2013), Mandal et al., Cell Stem Cell, Volume 15, Issue 5,p643-652, 6 Nov. 2014, Wang et al. (PLoS One. 2014 Dec. 26;9(12):e115987. doi: 10.1371/journal.pone.0115987), Fine et al. (Sci Rep.2015 Jul. 1; 5:10777. doi: 10.1038/srep10777) and Li et al. (J GenVirol. 2015 August; 96(8):2381-93. doi: 10.1099/vir.0.000139. Epub 2015Apr. 8) for targeting CCR5 with the CRISPR Cas system of the presentinvention.

X-linked Chronic granulomatous disease (CGD) is a hereditary disorder ofhost defense due to absent or decreased activity of phagocyte NADPHoxidase. Using a CRISPR-Cas9 system that targets and corrects themutation (absent or decreased activity of phagocyte NADPH oxidase)(e.g., with a suitable HDR template that delivers a coding sequence forphagocyte NADPH oxidase); specifically, the sgRNA can target mutationthat gives rise to CGD (deficient phagocyte NADPH oxidase), and the HDRcan provide coding for proper expression of phagocyte NADPH oxidase. AnsgRNA that targets the mutation-and-Cas9 protein containing particle iscontacted with HSCs carrying the mutation. The particle also can containa suitable HDR template to correct the mutation for proper expression ofphagocyte NADPH oxidase; or the HSC can be contacted with a secondparticle or a vector that contains or delivers the HDR template. The socontacted cells can be administered; and optionally treated/expanded;cf. Cartier.

Fanconi anemia: Mutations in at least 15 genes (FANCA, FANCB, FANCC,FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ/BACH1/BRIP1,FANCL/PHF9/POG, FANCM, FANCN/PALB2, FANCO/Rad51C, and FANCP/SLX4/BTBD12)can cause Fanconi anemia. Proteins produced from these genes areinvolved in a cell process known as the FA pathway. The FA pathway isturned on (activated) when the process of making new copies of DNA,called DNA replication, is blocked due to DNA damage. The FA pathwaysends certain proteins to the area of damage, which trigger DNA repairso DNA replication can continue. The FA pathway is particularlyresponsive to a certain type of DNA damage known as interstrandcross-links (ICLs). ICLs occur when two DNA building blocks(nucleotides) on opposite strands of DNA are abnormally attached orlinked together, which stops the process of DNA replication. ICLs can becaused by a buildup of toxic substances produced in the body or bytreatment with certain cancer therapy drugs. Eight proteins associatedwith Fanconi anemia group together to form a complex known as the FAcore complex. The FA core complex activates two proteins, called FANCD2and FANCI. The activation of these two proteins brings DNA repairproteins to the area of the ICL so the cross-link can be removed and DNAreplication can continue. the FA core complex. More in particular, theFA core complex is a nuclear multiprotein complex consisting of FANCA,FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM, functions as an E3ubiquitin ligase and mediates the activation of the ID complex, which isa heterodimer composed of FANCD2 and FANCI. Once monoubiquitinated, itinteracts with classical tumor suppressors downstream of the FA pathwayincluding FANCD1/BRCA2, FANCN/PALB2, FANCJ/BRIP1, and FANCO/Rad51C andthereby contributes to DNA repair via homologous recombination (HR).Eighty to 90 percent of FA cases are due to mutations in one of threegenes, FANCA, FANCC, and FANCG. These genes provide instructions forproducing components of the FA core complex. Mutations in such genesassociated with the FA core complex will cause the complex to benonfunctional and disrupt the entire FA pathway. As a result, DNA damageis not repaired efficiently and ICLs build up over time. Geiselhart,“Review Article, Disrupted Signaling through the Fanconi Anemia PathwayLeads to Dysfunctional Hematopoietic Stem Cell Biology: UnderlyingMechanisms and Potential Therapeutic Strategies,” Anemia Volume 2012(2012), Article ID 265790, http://dx.doi.org/10.1155/2012/265790discussed FA and an animal experiment involving intrafemoral injectionof a lentivirus encoding the FANCC gene resulting in correction of HSCsin vivo. Using a CRISPR-Cas9 system that targets and one or more of themutations associated with FA, for instance a CRISPR-Cas9 system havingsgRNA(s) and HDR template(s) that respectively targets one or more ofthe mutations of FANCA, FANCC, or FANCG that give rise to FA and providecorrective expression of one or more of FANCA, FANCC or FANCG; e.g., thesgRNA can target a mutation as to FANCC, and the HDR can provide codingfor proper expression of FANCC. An sgRNA that targets the mutation(s)(e.g., one or more involved in FA, such as mutation(s) as to any one ormore of FANCA, FANCC or FANCG)-and-Cas9 protein containing particle iscontacted with HSCs carrying the mutation(s). The particle also cancontain a suitable HDR template(s) to correct the mutation for properexpression of one or more of the proteins involved in FA, such as anyone or more of FANCA, FANCC or FANCG; or the HSC can be contacted with asecond particle or a vector that contains or delivers the HDR template.The so contacted cells can be administered; and optionallytreated/expanded; cf. Cartier.

The particle in the herein discussion (e.g., as to containing sgRNA(s)and Cas9, optionally HDR template(s), or HDR template(s); for instanceas to Hemophilia B, SCID, SCID-X1, ADA-SCID, Hereditary tyrosinemia,β-thalassemia, X-linked CGD, Wiskott-Aldrich syndrome, Fanconi anemia,adrenoleukodystrophy (ALD), metachromatic leukodystrophy (MLD),HIV/AIDS, Immunodeficiency disorder, Hematologic condition, or geneticlysosomal storage disease) is advantageously obtained or obtainable fromadmixing an sgRNA(s) and Cas9 protein mixture (optionally containing HDRtemplate(s) or such mixture only containing HDR template(s) whenseparate particles as to template(s) is desired) with a mixturecomprising or consisting essentially of or consisting of surfactant,phospholipid, biodegradable polymer, lipoprotein and alcohol (whereinone or more sgRNA targets the genetic locus or loci in the HSC).

Indeed, the invention is especially suited for treating hematopoieticgenetic disorders with genome editing, and immunodeficiency disorders,such as genetic immunodeficiency disorders, especially through using theparticle technology herein-discussed. Genetic immunodeficiencies arediseases where genome editing interventions of the instant invention cansuccessful. The reasons include: Hematopoietic cells, of which immunecells are a subset, are therapeutically accessible. They can be removedfrom the body and transplanted autologously or allogenically. Further,certain genetic immunodeficiencies, e.g., severe combinedimmunodeficiency (SCID), create a proliferative disadvantage for immunecells. Correction of genetic lesions causing SCID by rare, spontaneous‘reverse’ mutations indicates that correcting even one lymphocyteprogenitor may be sufficient to recover immune function in patients . .. / . . . / . . ./Users/t_kowalski/AppData/Local/Microsoft/Windows/Temporary InternetFiles/Content.Outlook/GA8VY8LK/Treating SCID for Ellen.docx—_ENREF_1 SeeBousso, P., et al. Diversity, functionality, and stability of the T cellrepertoire derived in vivo from a single human T cell precursor.Proceedings of the National Academy of Sciences of the United States ofAmerica 97, 274-278 (2000). The selective advantage for edited cellsallows for even low levels of editing to result in a therapeutic effect.This effect of the instant invention can be seen in SCID,Wiskott-Aldrich Syndrome, and the other conditions mentioned herein,including other genetic hematopoietic disorders such as alpha- andbeta-thalassemia, where hemoglobin deficiencies negatively affect thefitness of erythroid progenitors.

The activity of NHEJ and HDR DSB repair varies significantly by celltype and cell state. NHEJ is not highly regulated by the cell cycle andis efficient across cell types, allowing for high levels of genedisruption in accessible target cell populations. In contrast, HDR actsprimarily during S/G2 phase, and is therefore restricted to cells thatare actively dividing, limiting treatments that require precise genomemodifications to mitotic cells [Ciccia, A. & Elledge, S. J. Molecularcell 40, 179-204 (2010); Chapman, J. R., et al. Molecular cell 47,497-510 (2012)].

The efficiency of correction via HDR may be controlled by the epigeneticstate or sequence of the targeted locus, or the specific repair templateconfiguration (single vs. double stranded, long vs. short homology arms)used [Hacein-Bey-Abina, S., et al. The New England journal of medicine346, 1185-1193 (2002); Gaspar, H. B., et al. Lancet 364, 2181-2187(2004); Beumer, K. J., et al. G3 (2013)]. The relative activity of NHEJand HDR machineries in target cells may also affect gene correctionefficiency, as these pathways may compete to resolve DSBs [Beumer, K.J., et al. Proceedings of the National Academy of Sciences of the UnitedStates of America 105, 19821-19826 (2008)]. HDR also imposes a deliverychallenge not seen with NHEJ strategies, as it requires the concurrentdelivery of nucleases and repair templates. In practice, theseconstraints have so far led to low levels of HDR in therapeuticallyrelevant cell types. Clinical translation has therefore largely focusedon NHEJ strategies to treat disease, although proof-of-conceptpreclinical HDR treatments have now been described for mouse models ofhaemophilia B and hereditary tyrosinemia [Li, H., et al. Nature 475,217-221 (2011); Yin, H., et al. Nature biotechnology 32, 551-553(2014)].

Any given genome editing application may comprise combinations ofproteins, small RNA molecules, and/or repair templates, making deliveryof these multiple parts substantially more challenging than smallmolecule therapeutics. Two main strategies for delivery of genomeediting tools have been developed: ex vivo and in vivo. In ex vivotreatments, diseased cells are removed from the body, edited and thentransplanted back into the patient (FIG. 3, top panel). Ex vivo editinghas the advantage of allowing the target cell population to be welldefined and the specific dosage of therapeutic molecules delivered tocells to be specified. The latter consideration may be particularlyimportant when off-target modifications are a concern, as titrating theamount of nuclease may decrease such mutations (Hsu et al., 2013).Another advantage of ex vivo approaches is the typically high editingrates that can be achieved, due to the development of efficient deliverysystems for proteins and nucleic acids into cells in culture forresearch and gene therapy applications.

There may be drawbacks with ex vivo approaches that limit application toa small number of diseases. For instance, target cells must be capableof surviving manipulation outside the body. For many tissues, like thebrain, culturing cells outside the body is a major challenge, becausecells either fail to survive, or lose properties necessary for theirfunction in vivo. Thus, in view of this disclosure and the knowledge inthe art, ex vivo therapy as to tissues with adult stem cell populationsamenable to ex vivo culture and manipulation, such as the hematopoieticsystem, by the CRISPR-Cas9 system are enabled. [Bunn, H. F. & Aster, J.Pathophysiology of blood disorders, (McGraw-Hill, New York, 2011)]

In vivo genome editing involves direct delivery of editing systems tocell types in their native tissues (FIG. 3, bottom panels). In vivoediting allows diseases in which the affected cell population is notamenable to ex vivo manipulation to be treated. Furthermore, deliveringnucleases to cells in situ allows for the treatment of multiple tissueand cell types. These properties probably allow in vivo treatment to beapplied to a wider range of diseases than ex vivo therapies.

To date, in vivo editing has largely been achieved through the use ofviral vectors with defined, tissue-specific tropism. Such vectors arecurrently limited in terms of cargo carrying capacity and tropism,restricting this mode of therapy to organ systems where transductionwith clinically useful vectors is efficient, such as the liver, muscleand eye [Kotterman, M. A. & Schaffer, D. V. Nature reviews. Genetics 15,445-451 (2014); Nguyen, T. H. & Ferry, N. Gene therapy 11 Suppl 1,S76-84 (2004); Boye, S. E., et al. Molecular therapy: the journal of theAmerican Society of Gene Therapy 21, 509-519 (2013)].

A potential barrier for in vivo delivery is the immune response that maybe created in response to the large amounts of virus necessary fortreatment, but this phenomenon is not unique to genome editing and isobserved with other virus based gene therapies [Bessis, N., et al. Genetherapy 11 Suppl 1, S10-17 (2004)]. It is also possible that peptidesfrom editing nucleases themselves are presented on MHC Class I moleculesto stimulate an immune response, although there is little evidence tosupport this happening at the preclinical level. Another majordifficulty with this mode of therapy is controlling the distribution andconsequently the dosage of genome editing nucleases in vivo, leading tooff-target mutation profiles that may be difficult to predict. However,in view of this disclosure and the knowledge in the art, including theuse of virus- and particle-based therapies being used in the treatmentof cancers, in vivo modification of HSCs, for instance by delivery byeither particle or virus, is within the ambit of the the skilled person.

Ex Vivo Editing Therapy:

The long standing clinical expertise with the purification, culture andtransplantation of hematopoietic cells has made diseases affecting theblood system such as SCID, Fanconi anemia, Wiskott-Aldrich syndrome andsickle cell anemia the focus of ex vivo editing therapy. Another reasonto focus on hematopoietic cells is that, thanks to previous efforts todesign gene therapy for blood disorders, delivery systems of relativelyhigh efficiency already exist. With these advantages, this mode oftherapy can be applied to diseases where edited cells possess a fitnessadvantage, so that a small number of engrafted, edited cells can expandand treat disease. One such disease is HIV, where infection results in afitness disadvantage to CD4+ T cells. The rationale for genome editingfor HIV treatment originates from the observation that individualshomozygous for loss of function mutations in CCR5, a cellularco-receptor for the virus, are highly resistant to infection andotherwise healthy, suggesting that mimicking this mutation with genomeediting could be a safe and effective therapeutic strategy [Liu, R., etal. Cell 86, 367-377 (1996)]. This idea was clinically validated when anHIV infected patient was given an allogeneic bone marrow transplant froma donor homozygous for a loss of function CCR5 mutation, resulting inundetectable levels of HIV and restoration of normal CD4 T-cell counts[Hutter, G., et al. The New England journal of medicine 360, 692-698(2009)]. Although bone marrow transplantation is not a realistictreatment strategy for most HIV patients, due to cost and potentialgraft vs. host disease, HIV therapies that convert a patient's ownT-cells into CCR5 are.

Early studies using ZFNs and NHEJ to knockout CCR5 in humanized mousemodels of HIV showed that transplantation of CCR5 edited CD4 T cellsimproved viral load and CD4 T-cell counts [Perez, E. E., et al. Naturebiotechnology 26, 808-816 (2008)]. Importantly, these models also showedthat HIV infection resulted in selection for CCR5 null cells, suggestingthat editing confers a fitness advantage and potentially allowing asmall number of edited cells to create a therapeutic effect.

As a result of this and other promising preclinical studies, genomeediting therapy that knocks out CCR5 in patient T cells has now beentested in humans [Holt, N., et al. Nature biotechnology 28, 839-847(2010); Li, L., et al. Molecular therapy: the journal of the AmericanSociety of Gene Therapy 21, 1259-1269 (2013)]. In a recent phase Iclinical trial, CD4+ T cells from patients with HIV were removed, editedwith ZFNs designed to knockout the CCR5 gene, and autologouslytransplanted back into patients [Tebas, P., et al. The New Englandjournal of medicine 370, 901-910 (2014)]. Early results from this trialsuggest that genome editing through ZFNs of the CCR5 locus is safe,although the follow up time is too short to fully understand the risksand efficacy of treatment.

Ex vivo editing therapy has been recently extended to include genecorrection strategies. The barriers to HDR ex vivo were overcome in arecent paper from Genovese and colleagues, who achieved gene correctionof a mutated IL2RG gene in hematopoietic stem cells (HSCs) obtained froma patient suffering from SCID-X1 [Genovese, P., et al. Nature 510,235-240 (2014)]. Genovese et. al. accomplished gene correction in HSCsusing a multimodal strategy. First, HSCs were transduced usingintegration-deficient lentivirus containing an HDR template encoding atherapeutic cDNA for IL2RG. Following transduction, cells wereelectroporated with mRNA encoding ZFNs targeting a mutational hotspot inIL2RG to stimulate HDR based gene correction. To increase HDR rates,culture conditions were optimized with small molecules to encourage HSCdivision. With optimized culture conditions, nucleases and HDRtemplates, gene corrected HSCs from the SCID-X1 patient were obtained inculture at therapeutically relevant rates. HSCs from unaffectedindividuals that underwent the same gene correction procedure couldsustain long-term hematopoiesis in mice, the gold standard for HSCfunction. HSCs are capable of giving rise to all hematopoietic celltypes and can be autologously transplanted, making them an extremelyvaluable cell population for all hematopoietic genetic disorders[Weissman, I. L. & Shizuru, J. A. Blood 112, 3543-3553 (2008)]. Genecorrected HSCs could, in principle, be used to treat a wide range ofgenetic blood disorders making this study an exciting breakthrough fortherapeutic genome editing.

In Vivo Editing Therapy:

In vivo editing can be used advantageously from this disclosure and theknowledge in the art. For organ systems where delivery is efficient,there have already been a number of exciting preclinical therapeuticsuccesses. The first example of successful in vivo editing therapy wasdemonstrated in a mouse model of haemophilia B [Li, H., et al. Nature475, 217-221 (2011)]. As noted earlier, Haemophilia B is an X-linkedrecessive disorder caused by loss-of-function mutations in the geneencoding Factor IX, a crucial component of the clotting cascade.Recovering Factor IX activity to above 1% of its levels in severelyaffected individuals can transform the disease into a significantlymilder form, as infusion of recombinant Factor IX into such patientsprophylactically from a young age to achieve such levels largelyameliorates clinical complications [Lofqvist, T., et al. Journal ofinternal medicine 241, 395-400 (1997)]. Thus, only low levels of HDRgene correction are necessary to change clinical outcomes for patients.In addition, Factor IX is synthesized and secreted by the liver, anorgan that can be transduced efficiently by viral vectors encodingediting systems.

Using hepatotropic adeno-associated viral (AAV) serotypes encoding ZFNsand a corrective HDR template, up to 7% gene correction of a mutated,humanized Factor IX gene in the murine liver was achieved [Li, H., etal. Nature 475, 217-221 (2011)]. This resulted in improvement of clotformation kinetics, a measure of the function of the clotting cascade,demonstrating for the first time that in vivo editing therapy is notonly feasible, but also efficacious. As discussed herein, the skilledperson is positioned from the teachings herein and the knowledge in theart, e.g., Li to address Haemophilia B with a particle-containing HDRtemplate and a CRISPR-Cas9 system that targets the mutation of theX-linked recessive disorder to reverse the loss-of-function mutation.

Building on this study, other groups have recently used in vivo genomeediting of the liver with CRISPR-Cas9 to successfully treat a mousemodel of hereditary tyrosinemia and to create mutations that provideprotection against cardiovascular disease. These two distinctapplications demonstrate the versatility of this approach for disordersthat involve hepatic dysfunction [Yin, H., et al. Nature biotechnology32, 551-553 (2014); Ding, Q., et al. Circulation research 115, 488-492(2014)]. Application of in vivo editing to other organ systems arenecessary to prove that this strategy is widely applicable. Currently,efforts to optimize both viral and non-viral vectors are underway toexpand the range of disorders that can be treated with this mode oftherapy [Kotterman, M. A. & Schaffer, D. V. Nature reviews. Genetics 15,445-451 (2014); Yin, H., et al. Nature reviews. Genetics 15, 541-555(2014)]. As discussed herein, the skilled person is positioned from theteachings herein and the knowledge in the art, e.g., Yin to addresshereditary tyrosinemia with a particle-containing HDR template and aCRISPR-Cas9 system that targets the mutation.

Targeted Deletion, Therapeutic Applications:

Targeted deletion of genes may be preferred. Preferred are, therefore,genes involved in immunodeficiency disorder, hematologic condition, orgenetic lysosomal storage disease, e.g., Hemophilia B, SCID, SCID-X1,ADA-SCID, Hereditary tyrosinemia, β-thalassemia, X-linked CGD,Wiskott-Aldrich syndrome, Fanconi anemia, adrenoleukodystrophy (ALD),metachromatic leukodystrophy (MLD), HIV/AIDS, other metabolic disorders,genes encoding mis-folded proteins involved in diseases, genes leadingto loss-of-function involved in diseases; generally, mutations that canbe targeted in an HSC, using any herein-discussed delivery system, withthe particle system considered advantageous.

In the present invention, the immunogenicity of the CRISPR enzyme inparticular may be reduced following the approach first set out in Tangriet al with respect to erythropoietin and subsequently developed.Accordingly, directed evolution or rational design may be used to reducethe immunogenicity of the CRISPR enzyme (for instance a Cas9) in thehost species (human or other species).

Genome Editing:

The CRISPR/Cas9 systems of the present invention can be used to correctgenetic mutations that were previously attempted with limited successusing TALEN and ZFN and lentiviruses, including as herein discussed; seealso WO2013163628 A2, Genetic Correction of Mutated Genes, publishedapplication of Duke University; US Patent Publication No. 20130145487assigned to Cellectis.

Blood:

The present invention also contemplates delivering the CRISPR-Cas systemto the blood. The plasma exosomes of Wahlgren et al. (Nucleic AcidsResearch, 2012, Vol. 40, No. 17 e130) were previously described and maybe utilized to deliver the CRISPR Cas system to the blood. The CRISPRCas system of the present invention is also contemplated to treathemoglobinopathies, such as thalassemias and sickle cell disease. See,e.g., International Patent Publication No. WO 2013/126794 for potentialtargets that may be targeted by the CRISPR Cas system of the presentinvention. Target sequences identified in US Patent Publication Nos.20110225664, 20110091441, 20100229252, 20090271881 and 20090222937 maybe of interest as to the present invention.

The target polynucleotide of a CRISPR complex may include a number ofdisease-associated genes and polynucleotides as well as signalingbiochemical pathway-associated genes. Examples of target polynucleotidesinclude a sequence associated with a signaling biochemical pathway,e.g., a signaling biochemical pathway-associated gene or polynucleotide.Examples of target polynucleotides include a disease associated gene orpolynucleotide. A “disease-associated” gene or polynucleotide refers toany gene or polynucleotide which is yielding transcription ortranslation products at an abnormal level or in an abnormal form incells derived from a disease-affected tissues compared with tissues orcells of a non disease control. It may be a gene that becomes expressedat an abnormally high level; it may be a gene that becomes expressed atan abnormally low level, where the altered expression correlates withthe occurrence and/or progression of the disease. A disease-associatedgene also refers to a gene possessing mutation(s) or genetic variationthat is directly responsible or is in linkage disequilibrium with agene(s) that is responsible for the etiology of a disease. Thetranscribed or translated products may be known or unknown, and may beat a normal or abnormal level. The target polynucleotide of a CRISPRcomplex can be any polynucleotide endogenous or exogenous to theeukaryotic cell. For example, the target polynucleotide can be apolynucleotide residing in the nucleus of the eukaryotic cell. Thetarget polynucleotide can be a sequence coding a gene product (e.g., aprotein) or a non-coding sequence (e.g., a regulatory polynucleotide ora junk DNA). The tareget can be a control element or a regulatoryelement or a promoter or an enhancer or a silencer. The promoter may, insome embodiments, be in the region of +200 bp or even +1000 bp from theTTS. In some embodiments, the regulatory region may be an enhancer. Theenhancer is typically more than +1000 bp from the TTS. More inparticular, expression of eukaryotic protein-coding genes generally isregulated through multiple cis-acting transcription-control regions.Some control elements are located close to the start site(promoter-proximal elements), whereas others lie more distant (enhancersand silencers) Promoters determine the site of transcription initiationand direct binding of RNA polymerase II. Three types of promotersequences have been identified in eukaryotic DNA. The TATA box, the mostcommon, is prevalent in rapidly transcribed genes. Initiator promotersinfrequently are found in some genes, and CpG islands are characteristicof transcribed genes. Promoter-proximal elements occur within ≈200 basepairs of the start site. Several such elements, containing up to ≈20base pairs, may help regulate a particular gene. Enhancers, which areusually ≈100-200 base pairs in length, contain multiple 8- to 20-bpcontrol elements. They may be located from 200 base pairs to tens ofkilobases upstream or downstream from a promoter, within an intron, ordownstream from the final exon of a gene. Promoter-proximal elements andenhancers may be cell-type specific, functioning only in specificdifferentiated cell types. However, any of these regions can be thetarget sequence and are encompassed by the concept that the tareget canbe a control element or a regulatory element or a promoter or anenhancer or a silencer. Without wishing to be bound by theory, it isbelieved that the target sequence should be associated with a PAM(protospacer adjacent motif); that is, a short sequence recognized bythe CRISPR complex. The precise sequence and length requirements for thePAM differ depending on the CRISPR enzyme used, but PAMs are typically2-5 base pair sequences adjacent the protospacer (that is, the targetsequence) Examples of PAM sequences are given in the examples sectionbelow, and the skilled person will be able to identify further PAMsequences for use with a given CRISPR enzyme.

The Efficiency of HDR:

Although the amount of genome modification in a target cell populationrequired to create a therapeutic effect differs depending on thedisease, the efficacy of most editing treatments are improved withincreased editing rates. As previously noted, editing rates arecontrolled by the activity of DSB repair pathways and the efficiency ofdelivery to cells of interest. Therefore improvements to either one ofthese factors are likely to improve the efficacy of editing treatments.Attempts to increase the activity rates of DSB repair pathways havelargely focused on HDR, as cell cycle regulation and the challenge ofdelivering an HDR template with nucleases makes strategies employingthis pathway less efficient than NHEJ. Cell cycle regulation has nowbeen somewhat by-passed for slowly cycling cell types throughstimulation of mitosis with pharmacologic agents ex vivo [Kormann, M.S., et al. Nature biotechnology 29, 154-157 (2011)]. However, trulypost-mitotic cells are unlikely to be amenable to such manipulation,limiting the applicability of this strategy. Attempts have been made tocompletely circumvent the need for HDR through direct ligation of DNAtemplates containing therapeutic transgenes into targeted DSBs. Suchligation events have been observed, but the rates are too low to beuseful for therapy [Ran, F. A., et al. Cell 154, 1380-1389 (2013);Orlando, S. J., et al. Nucleic acids research 38, e152 (2010)]. Likelydramatically new approaches are necessary to improve HDR efficiency andincrease the therapeutic efficacy of strategies requiring precisegenomic correction.

Barcoding:

In an aspect of the invention, barcoding techniques, e.g., ofWO/2013/138585 A1, can be adapted or integrated into the practice of theinvention. In an aspect of the invention, barcoding techniques ofWO/2013/138585 A1 can be adapted or integrated into the practice of theinvention. WO/2013/138585 A1 provides methods for simultaneouslydetermining the effect of a test condition on viability or proliferationof each of a plurality of genetically heterogeneous cell types. Themethods include: providing a unitary sample comprising a plurality of,e.g., five, ten, twenty, twenty-five, or more, genetically heterogeneouscell types (each individual cell type is genetically homogeneous withinitself, but differs from the others in the plurality), wherein each celltype further comprises: (i) an exogenous nucleic acid tag stablyintegrated into the genome of the cells, e.g., a tag comprising a coresequence that is unique to each cell type, and flanking amplificationprimer binding sequences that are the same in all of the cells of theplurality, and (ii) optionally, a marker, e.g., a selectable ordetectable marker; and a known number of cells of each cell type ispresent in the sample; exposing the sample to a test condition for aselected time; and detecting a level of the exogenous nucleic acid tagin each cell type, wherein the level of the exogenous nucleic acid tagis proportional to the number of living cells in the sample afterexposure to the test condition; and comparing the number of living cellsin the sample after exposure to the test condition to a reference numberof cells. The number of living cells in the sample after exposure to thetest condition as compared to the reference number of cells indicatesthe effect of the test condition on viability or proliferation of eachcell type. WO/2013/138585 A1 also provides methods for simultaneouslydetermining the effect of a test condition on viability or proliferationof each of a plurality of genetically heterogeneous cell types, whereinthe methods include providing a unitary sample comprising a pluralityof, e.g., five, ten, twenty, twenty-five, or more, geneticallyheterogeneous cell types, wherein each cell type further comprises: (i)an exogenous nucleic acid tag stably integrated into the genome of thecells, e.g., comprising a core sequence that is unique to each celltype, and flanking amplification primer binding sequences that are thesame in all of the cells of the plurality, and (ii) optionally, aselectable or detectable marker; and a known number of cells of eachcell type is present in the sample; implanting the sample into a livinganimal; exposing the sample to a test condition for a selected time;harvesting the sample from the animal; and detecting a level of theexogenous nucleic acid tag in each cell type of the sample, wherein thelevel of the exogenous nucleic acid tag correlates to the number ofliving cells in the sample after exposure to the test condition; andcomparing the number of living cells in the sample after exposure to thetest condition to a reference number of cells. The number of livingcells in the sample after exposure to the test condition as compared tothe reference number of cells indicates the effect of the test conditionon viability or proliferation of each cell type. The tag can be Cas9 oranother TAG or marker that is integrated into the genome of cells to betransplanted into or onto a non-human eukaryote, e.g., animal model, orthat is integrated into the genome of the non-human transgeniceukaryote, e.g., animal, mammal, primate, rodent, mouse, rat, rabbit,etc (along with coding for Cas9). The test condition can be theadministration or delivery of the RNA(s) to guide the Cas9 to induce oneor more or a plurality, e.g., 3-50 or more, mutations. The testcondition can be the administration, delivery or contacting with aputative chemical agent treatment and/or gene therapy treatment. The tagcan also be the one or more or a plurality, e.g., 3-50 or moremutations, and the test condition can be the administration, delivery orcontacting with a putative chemical agent treatment and/or gene therapytreatment.

Nucleic Acids, Amino Acids and Proteins, Regulatory Sequences, Vectors,Etc

Nucleic acids, amino acids and proteins: The invention uses nucleicacids to bind target DNA sequences. This is advantageous as nucleicacids are much easier and cheaper to produce than proteins, and thespecificity can be varied according to the length of the stretch wherehomology is sought. Complex 3-D positioning of multiple fingers, forexample is not required. The terms “polynucleotide”, “nucleotide”,“nucleotide sequence”, “nucleic acid” and “oligonucleotide” are usedinterchangeably. They refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Polynucleotides may have any three dimensional structure, andmay perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: coding or non-coding regionsof a gene or gene fragment, loci (locus) defined from linkage analysis,exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, shortinterfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA),ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. The term also encompassesnucleic-acid-like structures with synthetic backbones, see, e.g.,Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97/03211; WO96/39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. Apolynucleotide may comprise one or more modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms. A “wild type” can be a base line. As used herein the term“variant” should be taken to mean the exhibition of qualities that havea pattern that deviates from what occurs in nature. The terms“non-naturally occurring” or “engineered” are used interchangeably andindicate the involvement of the hand of man. The terms, when referringto nucleic acid molecules or polypeptides mean that the nucleic acidmolecule or the polypeptide is at least substantially free from at leastone other component with which they are naturally associated in natureand as found in nature. “Complementarity” refers to the ability of anucleic acid to form hydrogen bond(s) with another nucleic acid sequenceby either traditional Watson-Crick base pairing or other non-traditionaltypes. A percent complementarity indicates the percentage of residues ina nucleic acid molecule which can form hydrogen bonds (e.g.,Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5,6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%complementary). “Perfectly complementary” means that all the contiguousresidues of a nucleic acid sequence will hydrogen bond with the samenumber of contiguous residues in a second nucleic acid sequence.“Substantially complementary” as used herein refers to a degree ofcomplementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or morenucleotides, or refers to two nucleic acids that hybridize understringent conditions. As used herein, “stringent conditions” forhybridization refer to conditions under which a nucleic acid havingcomplementarity to a target sequence predominantly hybridizes with thetarget sequence, and substantially does not hybridize to non-targetsequences. Stringent conditions are generally sequence-dependent, andvary depending on a number of factors. In general, the longer thesequence, the higher the temperature at which the sequence specificallyhybridizes to its target sequence. Non-limiting examples of stringentconditions are described in detail in Tijssen (1993), LaboratoryTechniques In Biochemistry And Molecular Biology-Hybridization WithNucleic Acid Probes Part I, Second Chapter “Overview of principles ofhybridization and the strategy of nucleic acid probe assay”, Elsevier,N.Y. Where reference is made to a polynucleotide sequence, thencomplementary or partially complementary sequences are also envisaged.These are preferably capable of hybridising to the reference sequenceunder highly stringent conditions. Generally, in order to maximize thehybridization rate, relatively low-stringency hybridization conditionsare selected: about 20 to 25° C. lower than the thermal melting point(T_(m)). The T_(m) is the temperature at which 50% of specific targetsequence hybridizes to a perfectly complementary probe in solution at adefined ionic strength and pH. Generally, in order to require at leastabout 85% nucleotide complementarity of hybridized sequences, highlystringent washing conditions are selected to be about 5 to 15° C. lowerthan the T_(m). In order to require at least about 70% nucleotidecomplementarity of hybridized sequences, moderately-stringent washingconditions are selected to be about 15 to 30° C. lower than the T_(m).Highly permissive (very low stringency) washing conditions may be as lowas 50° C. below the T_(m), allowing a high level of mis-matching betweenhybridized sequences. Those skilled in the art will recognize that otherphysical and chemical parameters in the hybridization and wash stagescan also be altered to affect the outcome of a detectable hybridizationsignal from a specific level of homology between target and probesequences. Preferred highly stringent conditions comprise incubation in50% formamide, 5×SSC, and 1% SDS at 42° C., or incubation in 5×SSC and1% SDS at 65° C., with wash in 0.2×SSC and 0.1% SDS at 65° C.“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson Crick base pairing, Hoogstein binding, or inany other sequence specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combinationof these. A hybridization reaction may constitute a step in a moreextensive process, such as the initiation of PCR, or the cleavage of apolynucleotide by an enzyme. A sequence capable of hybridizing with agiven sequence is referred to as the “complement” of the given sequence.As used herein, the term “genomic locus” or “locus” (plural loci) is thespecific location of a gene or DNA sequence on a chromosome. A “gene”refers to stretches of DNA or RNA that encode a polypeptide or an RNAchain that has functional role to play in an organism and hence is themolecular unit of heredity in living organisms. For the purpose of thisinvention it may be considered that genes include regions which regulatethe production of the gene product, whether or not such regulatorysequences are adjacent to coding and/or transcribed sequences.Accordingly, a gene includes, but is not necessarily limited to,promoter sequences, terminators, translational regulatory sequences suchas ribosome binding sites and internal ribosome entry sites, enhancers,silencers, insulators, boundary elements, replication origins, matrixattachment sites and locus control regions. As used herein, “expressionof a genomic locus” or “gene expression” is the process by whichinformation from a gene is used in the synthesis of a functional geneproduct. The products of gene expression are often proteins, but innon-protein coding genes such as rRNA genes or tRNA genes, the productis functional RNA. The process of gene expression is used by all knownlife—eukaryotes (including multicellular organisms), prokaryotes(bacteria and archaea) and viruses to generate functional products tosurvive. As used herein “expression” of a gene or nucleic acidencompasses not only cellular gene expression, but also thetranscription and translation of nucleic acid(s) in cloning systems andin any other context. As used herein, “expression” also refers to theprocess by which a polynucleotide is transcribed from a DNA template(such as into and mRNA or other RNA transcript) and/or the process bywhich a transcribed mRNA is subsequently translated into peptides,polypeptides, or proteins. Transcripts and encoded polypeptides may becollectively referred to as “gene product.” If the polynucleotide isderived from genomic DNA, expression may include splicing of the mRNA ina eukaryotic cell. The terms “polypeptide”, “peptide” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non amino acids. The termsalso encompass an amino acid polymer that has been modified; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component. As used herein the term “aminoacid” includes natural and/or unnatural or synthetic amino acids,including glycine and both the D or L optical isomers, and amino acidanalogs and peptidomimetics. As used herein, the term “domain” or“protein domain” refers to a part of a protein sequence that may existand function independently of the rest of the protein chain. Asdescribed in aspects of the invention, sequence identity is related tosequence homology. Homology comparisons may be conducted by eye, or moreusually, with the aid of readily available sequence comparison programs.These commercially available computer programs may calculate percent (%)homology between two or more sequences and may also calculate thesequence identity shared by two or more amino acid or nucleic acidsequences. In some preferred embodiments, the capping region of thedTALEs described herein have sequences that are at least 95% identicalor share identity to the capping region amino acid sequences providedherein. Sequence homologies may be generated by any of a number ofcomputer programs known in the art, for example BLAST or FASTA, etc. Asuitable computer program for carrying out such an alignment is the GCGWisconsin Bestfit package (University of Wisconsin, U.S.A; Devereux etal., 1984, Nucleic Acids Research 12:387). Examples of other softwarethan may perform sequence comparisons include, but are not limited to,the BLAST package (see Ausubel et al., 1999 ibid—Chapter 18), FASTA(Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suiteof comparison tools. Both BLAST and FASTA are available for offline andonline searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60).However it is preferred to use the GCG Bestfit program. Percentage (%)sequence homology may be calculated over contiguous sequences, i.e., onesequence is aligned with the other sequence and each amino acid ornucleotide in one sequence is directly compared with the correspondingamino acid or nucleotide in the other sequence, one residue at a time.This is called an “ungapped” alignment. Typically, such ungappedalignments are performed only over a relatively short number ofresidues. Although this is a very simple and consistent method, it failsto take into consideration that, for example, in an otherwise identicalpair of sequences, one insertion or deletion may cause the followingamino acid residues to be put out of alignment, thus potentiallyresulting in a large reduction in % homology when a global alignment isperformed. Consequently, most sequence comparison methods are designedto produce optimal alignments that take into consideration possibleinsertions and deletions without unduly penalizing the overall homologyor identity score. This is achieved by inserting “gaps” in the sequencealignment to try to maximize local homology or identity. However, thesemore complex methods assign “gap penalties” to each gap that occurs inthe alignment so that, for the same number of identical amino acids, asequence alignment with as few gaps as possible—reflecting higherrelatedness between the two compared sequences—may achieve a higherscore than one with many gaps. “Affinity gap costs” are typically usedthat charge a relatively high cost for the existence of a gap and asmaller penalty for each subsequent residue in the gap. This is the mostcommonly used gap scoring system. High gap penalties may, of course,produce optimized alignments with fewer gaps. Most alignment programsallow the gap penalties to be modified. However, it is preferred to usethe default values when using such software for sequence comparisons.For example, when using the GCG Wisconsin Bestfit package the defaultgap penalty for amino acid sequences is −12 for a gap and −4 for eachextension. Calculation of maximum % homology therefore first requiresthe production of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (Devereux et al., 1984Nuc. Acids Research 12 p387). Examples of other software than mayperform sequence comparisons include, but are not limited to, the BLASTpackage (see Ausubel et al., 1999 Short Protocols in Molecular Biology,4^(th) Ed.—Chapter 18), FASTA (Altschul et al., 1990 J. Mol. Biol.403-410) and the GENEWORKS suite of comparison tools. Both BLAST andFASTA are available for offline and online searching (see Ausubel etal., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60).However, for some applications, it is preferred to use the GCG Bestfitprogram. A new tool, called BLAST 2 Sequences is also available forcomparing protein and nucleotide sequences (see FEMS Microbiol Lett.1999 174(2): 247-50; FEMS Microbiol Lett. 1999 177(1): 187-8 and thewebsite of the National Center for Biotechnology information at thewebsite of the National Institutes for Health). Although the final %homology may be measured in terms of identity, the alignment processitself is typically not based on an all-or-nothing pair comparison.Instead, a scaled similarity score matrix is generally used that assignsscores to each pair-wise comparison based on chemical similarity orevolutionary distance. An example of such a matrix commonly used is theBLOSUM62 matrix—the default matrix for the BLAST suite of programs. GCGWisconsin programs generally use either the public default values or acustom symbol comparison table, if supplied (see user manual for furtherdetails). For some applications, it is preferred to use the publicdefault values for the GCG package, or in the case of other software,the default matrix, such as BLOSUM62. Alternatively, percentagehomologies may be calculated using the multiple alignment feature inDNASIS™ (Hitachi Software), based on an algorithm, analogous to CLUSTAL(Higgins D G & Sharp P M (1988), Gene 73(1), 237-244). Once the softwarehas produced an optimal alignment, it is possible to calculate %homology, preferably % sequence identity. The software typically doesthis as part of the sequence comparison and generates a numericalresult. The sequences may also have deletions, insertions orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent substance. Deliberate amino acidsubstitutions may be made on the basis of similarity in amino acidproperties (such as polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues) and it istherefore useful to group amino acids together in functional groups.Amino acids may be grouped together based on the properties of theirside chains alone. However, it is more useful to include mutation dataas well. The sets of amino acids thus derived are likely to be conservedfor structural reasons. These sets may be described in the form of aVenn diagram (Livingstone C. D. and Barton G. J. (1993) “Proteinsequence alignments: a strategy for the hierarchical analysis of residueconservation” Comput. Appl. Biosci. 9: 745-756) (Taylor W. R. (1986)“The classification of amino acid conservation” J. Theor. Biol. 119;205-218). Conservative substitutions may be made, for example accordingto the table below which describes a generally accepted Venn diagramgrouping of amino acids.

Set Sub-set Hydrophobic F W Y H K M I L V A G C Aromatic F W Y HAliphatic I L V Polar W Y H K R E D C S T N Q Charged H K R E DPositively charged H K R Negatively charged E D Small V C A G S P T N DTiny A G S

Embodiments of the invention include sequences (both polynucleotide orpolypeptide) which may comprise homologous substitution (substitutionand replacement are both used herein to mean the interchange of anexisting amino acid residue or nucleotide, with an alternative residueor nucleotide) that may occur i.e., like-for-like substitution in thecase of amino acids such as basic for basic, acidic for acidic, polarfor polar, etc. Non-homologous substitution may also occur i.e., fromone class of residue to another or alternatively involving the inclusionof unnatural amino acids such as ornithine (hereinafter referred to asZ), diaminobutyric acid ornithine (hereinafter referred to as B),norleucine ornithine (hereinafter referred to as O), pyriylalanine,thienylalanine, naphthylalanine and phenylglycine. Variant amino acidsequences may include suitable spacer groups that may be insertedbetween any two amino acid residues of the sequence including alkylgroups such as methyl, ethyl or propyl groups in addition to amino acidspacers such as glycine or β-alanine residues. A further form ofvariation, which involves the presence of one or more amino acidresidues in peptoid form, may be well understood by those skilled in theart. For the avoidance of doubt, “the peptoid form” is used to refer tovariant amino acid residues wherein the α-carbon substituent group is onthe residue's nitrogen atom rather than the α-carbon. Processes forpreparing peptides in the peptoid form are known in the art, for exampleSimon R J et al., PNAS (1992) 89(20), 9367-9371 and Horwell D C, TrendsBiotechnol. (1995) 13(4), 132-134.

For purpose of this invention, amplification means any method employinga primer and a polymerase capable of replicating a target sequence withreasonable fidelity. Amplification may be carried out by natural orrecombinant DNA polymerases such as TaqGold™, T7 DNA polymerase, Klenowfragment of E. coli DNA polymerase, and reverse transcriptase. Apreferred amplification method is PCR.

In certain aspects the invention involves vectors. A used herein, a“vector” is a tool that allows or facilitates the transfer of an entityfrom one environment to another. It is a replicon, such as a plasmid,phage, or cosmid, into which another DNA segment may be inserted so asto bring about the replication of the inserted segment. Generally, avector is capable of replication when associated with the proper controlelements. In general, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. Vectors include, but are not limited to, nucleic acidmolecules that are single-stranded, double-stranded, or partiallydouble-stranded; nucleic acid molecules that comprise one or more freeends, no free ends (e.g. circular); nucleic acid molecules that compriseDNA, RNA, or both; and other varieties of polynucleotides known in theart. One type of vector is a “plasmid,” which refers to a circulardouble stranded DNA loop into which additional DNA segments can beinserted, such as by standard molecular cloning techniques. Another typeof vector is a viral vector, wherein virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.retroviruses, replication defective retroviruses, adenoviruses,replication defective adenoviruses, and adeno-associated viruses(AAVs)). Viral vectors also include polynucleotides carried by a virusfor transfection into a host cell. Certain vectors are capable ofautonomous replication in a host cell into which they are introduced(e.g. bacterial vectors having a bacterial origin of replication andepisomal mammalian vectors). Other vectors (e.g., non-episomal mammalianvectors) are integrated into the genome of a host cell upon introductioninto the host cell, and thereby are replicated along with the hostgenome. Moreover, certain vectors are capable of directing theexpression of genes to which they are operatively-linked. Such vectorsare referred to herein as “expression vectors.” Common expressionvectors of utility in recombinant DNA techniques are often in the formof plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). With regards torecombination and cloning methods, mention is made of U.S. patentapplication Ser. No. 10/815,730, published Sep. 2, 2004 as US2004-0171156 A1, the contents of which are herein incorporated byreference in their entirety.

Aspects of the invention relate to bicistronic vectors for chimeric RNAand Cas9. Bicistronic expression vectors for chimeric RNA and Cas9 arepreferred. In general and particularly in this embodiment Cas9 ispreferably driven by the CBh promoter. The chimeric RNA may preferablybe driven by a Pol III promoter, such as a U6 promoter. Ideally the twoare combined. The chimeric guide RNA typically consists of a 20 bp guidesequence (Ns) and this may be joined to the tracr sequence (running fromthe first “U” of the lower strand to the end of the transcript). Thetracr sequence may be truncated at various positions as indicated. Theguide and tracr sequences are separated by the tracr-mate sequence,which may be GUUUUAGAGCUA (SEQ ID NO: 38). This may be followed by theloop sequence GAAA as shown. Both of these are preferred examples.Applicants have demonstrated Cas9-mediated indels at the human EMX1 andPVALB loci by SURVEYOR assays. ChiRNAs are indicated by their “+n”designation, and crRNA refers to a hybrid RNA where guide and tracrsequences are expressed as separate transcripts. Throughout thisapplication, chimeric RNA may also be called single guide, or syntheticguide RNA (sgRNA). The loop is preferably GAAA, but it is not limited tothis sequence or indeed to being only 4 bp in length. Indeed, preferredloop forming sequences for use in hairpin structures are fournucleotides in length, and most preferably have the sequence GAAA.However, longer or shorter loop sequences may be used, as mayalternative sequences. The sequences preferably include a nucleotidetriplet (for example, AAA), and an additional nucleotide (for example Cor G). Examples of loop forming sequences include CAAA and AAAG. Inpracticing any of the methods disclosed herein, a suitable vector can beintroduced to a cell or an embryo via one or more methods known in theart, including without limitation, microinjection, electroporation,sonoporation, biolistics, calcium phosphate-mediated transfection,cationic transfection, liposome transfection, dendrimer transfection,heat shock transfection, nucleofection transfection, magnetofection,lipofection, impalefection, optical transfection, proprietaryagent-enhanced uptake of nucleic acids, and delivery via liposomes,immunoliposomes, virosomes, or artificial virions. In some methods, thevector is introduced into an embryo by microinjection. The vector orvectors may be microinjected into the nucleus or the cytoplasm of theembryo. In some methods, the vector or vectors may be introduced into acell by nucleofection.

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g. transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g. liver,pancreas), or particular cell types (e.g. lymphocytes). Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g. 1, 2,3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g.1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters(e.g. 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof.Examples of pol III promoters include, but are not limited to, U6 and H1promoters. Examples of pol II promoters include, but are not limited to,the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally withthe RSV enhancer), the cytomegalovirus (CMV) promoter (optionally withthe CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)],the SV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EG1αpromoter. Also encompassed by the term “regulatory element” are enhancerelements, such as WPRE; CMV enhancers; the R-U5′ segment in LTR ofHTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer;and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc.Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It will beappreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression desired, etc. A vectorcan be introduced into host cells to thereby produce transcripts,proteins, or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., clustered regularlyinterspersed short palindromic repeats (CRISPR) transcripts, proteins,enzymes, mutant forms thereof, fusion proteins thereof, etc.). Withregards to regulatory sequences, mention is made of U.S. patentapplication Ser. No. 10/491,026, the contents of which are incorporatedby reference herein in their entirety. With regards to promoters,mention is made of PCT publication WO 2011/028929 and U.S. applicationSer. No. 12/511,940, the contents of which are incorporated by referenceherein in their entirety.

Vectors can be designed for expression of CRISPR transcripts (e.g.nucleic acid transcripts, proteins, or enzymes) in prokaryotic oreukaryotic cells. For example, CRISPR transcripts can be expressed inbacterial cells such as Escherichia coli, insect cells (usingbaculovirus expression vectors), yeast cells, or mammalian cells.Suitable host cells are discussed further in Goeddel, GENE EXPRESSIONTECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.(1990). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Vectors may be introduced and propagated in a prokaryote or prokaryoticcell. In some embodiments, a prokaryote is used to amplify copies of avector to be introduced into a eukaryotic cell or as an intermediatevector in the production of a vector to be introduced into a eukaryoticcell (e.g. amplifying a plasmid as part of a viral vector packagingsystem). In some embodiments, a prokaryote is used to amplify copies ofa vector and express one or more nucleic acids, such as to provide asource of one or more proteins for delivery to a host cell or hostorganism. Expression of proteins in prokaryotes is most often carriedout in Escherichia coli with vectors containing constitutive orinducible promoters directing the expression of either fusion ornon-fusion proteins. Fusion vectors add a number of amino acids to aprotein encoded therein, such as to the amino terminus of therecombinant protein. Such fusion vectors may serve one or more purposes,such as: (i) to increase expression of recombinant protein; (ii) toincrease the solubility of the recombinant protein; and (iii) to aid inthe purification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Example fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amrann etal., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENEEXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, SanDiego, Calif. (1990) 60-89). In some embodiments, a vector is a yeastexpression vector. Examples of vectors for expression in yeastSaccharomyces cerivisae include pYepSec1 (Baldari, et al., 1987. EMBO J.6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30: 933-943),pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (InvitrogenCorporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego,Calif.). In some embodiments, a vector drives protein expression ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., SF9cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

In some embodiments, a vector is capable of driving expression of one ormore sequences in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, 1987.Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195).When used in mammalian cells, the expression vector's control functionsare typically provided by one or more regulatory elements. For example,commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus, simian virus 40, and others disclosed herein and knownin the art. For other suitable expression systems for both prokaryoticand eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

In some embodiments, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) andimmunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the α-fetoprotein promoter (Campes andTilghman, 1989. Genes Dev. 3: 537-546). With regards to theseprokaryotic and eukaryotic vectors, mention is made of U.S. Pat. No.6,750,059, the contents of which are incorporated by reference herein intheir entirety. Other embodiments of the invention may relate to the useof viral vectors, with regards to which mention is made of U.S. patentapplication Ser. No. 13/092,085, the contents of which are incorporatedby reference herein in their entirety. Tissue-specific regulatoryelements are known in the art and in this regard, mention is made ofU.S. Pat. No. 7,776,321, the contents of which are incorporated byreference herein in their entirety. In some embodiments, a regulatoryelement is operably linked to one or more elements of a CRISPR system soas to drive expression of the one or more elements of the CRISPR system.In general, CRISPRs (Clustered Regularly Interspaced Short PalindromicRepeats), also known as SPIDRs (SPacer Interspersed Direct Repeats),constitute a family of DNA loci that are usually specific to aparticular bacterial species. The CRISPR locus comprises a distinctclass of interspersed short sequence repeats (SSRs) that were recognizedin E. coli (Ishino et al., J. Bacteriol., 169:5429-5433 [1987]; andNakata et al., J. Bacteriol., 171:3553-3556 [1989]), and associatedgenes. Similar interspersed SSRs have been identified in Haloferaxmediterranei, Streptococcus pyogenes, Anabaena, and Mycobacteriumtuberculosis (See, Groenen et al., Mol. Microbiol., 10:1057-1065 [1993];Hoe et al., Emerg. Infect. Dis., 5:254-263 [1999]; Masepohl et al.,Biochim. Biophys. Acta 1307:26-30 [1996]; and Mojica et al., Mol.Microbiol., 17:85-93 [1995]). The CRISPR loci typically differ fromother SSRs by the structure of the repeats, which have been termed shortregularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ. Biol.,6:23-33 [2002]; and Mojica et al., Mol. Microbiol., 36:244-246 [2000]).In general, the repeats are short elements that occur in clusters thatare regularly spaced by unique intervening sequences with asubstantially constant length (Mojica et al., [2000], supra). Althoughthe repeat sequences are highly conserved between strains, the number ofinterspersed repeats and the sequences of the spacer regions typicallydiffer from strain to strain (van Embden et al., J. Bacteriol.,182:2393-2401 [2000]). CRISPR loci have been identified in more than 40prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575[2002]; and Mojica et al., [2005]) including, but not limited toAeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula,Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus,Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium,Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus,Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma,Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas,Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella,Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus,Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia,Treponema, and Thermotoga.

In some embodiments, the CRISPR enzyme is part of a fusion proteincomprising one or more heterologous protein domains (e.g. about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition tothe CRISPR enzyme). A CRISPR enzyme fusion protein may comprise anyadditional protein sequence, and optionally a linker sequence betweenany two domains. Examples of protein domains that may be fused to aCRISPR enzyme include, without limitation, epitope tags, reporter genesequences, and protein domains having one or more of the followingactivities: methylase activity, demethylase activity, transcriptionactivation activity, transcription repression activity, transcriptionrelease factor activity, histone modification activity, RNA cleavageactivity and nucleic acid binding activity. Non-limiting examples ofepitope tags include histidine (His) tags, V5 tags, FLAG tags, influenzahemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx)tags. Examples of reporter genes include, but are not limited to,glutathione-S-transferase (GST), horseradish peroxidase (HRP),chloramphenicol acetyltransferase (CAT) beta-galactosidase,beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed,DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP),and autofluorescent proteins including blue fluorescent protein (BFP). ACRISPR enzyme may be fused to a gene sequence encoding a protein or afragment of a protein that bind DNA molecules or bind other cellularmolecules, including but not limited to maltose binding protein (MBP),S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domainfusions, and herpes simplex virus (HSV) BP16 protein fusions. Additionaldomains that may form part of a fusion protein comprising a CRISPRenzyme are described in US20110059502, incorporated herein by reference.In some embodiments, a tagged CRISPR enzyme is used to identify thelocation of a target sequence.

In some embodiments, a CRISPR enzyme may form a component of aninducible system. The inducible nature of the system would allow forspatiotemporal control of gene editing or gene expression using a formof energy. The form of energy may include but is not limited toelectromagnetic radiation, sound energy, chemical energy and thermalenergy. Examples of inducible system include tetracycline induciblepromoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptionactivations systems (FKBP, ABA, etc), or light inducible systems(Phytochrome, LOV domains, or cryptochrome), In one embodiment, theCRISPR enzyme may be a part of a Light Inducible TranscriptionalEffector (LITE) to direct changes in transcriptional activity in asequence-specific manner. The components of a light may include a CRISPRenzyme, a light-responsive cytochrome heterodimer (e.g. from Arabidopsisthaliana), and a transcriptional activation/repression domain. Furtherexamples of inducible DNA binding proteins and methods for their use areprovided in U.S. 61/736,465 and U.S. 61/721,283, which is herebyincorporated by reference in its entirety.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, molecular biology, microbiology, cell biology, genomics andrecombinant DNA, which are within the skill of the art. See Sambrook,Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2ndedition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel,et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press,Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, ALABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).

Modifying a Target

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or invitro. In some embodiments, the method comprises sampling a cell orpopulation of cells from a human or non-human animal, and modifying thecell or cells. Culturing may occur at any stage ex vivo. The cell orcells may even be re-introduced into the non-human animal or plant. Forre-introduced cells it is particularly preferred that the cells are stemcells.

In some embodiments, the method comprises allowing a CRISPR complex tobind to the target polynucleotide to effect cleavage of said targetpolynucleotide thereby modifying the target polynucleotide, wherein theCRISPR complex comprises a CRISPR enzyme complexed with a guide sequencehybridized or hybridizable to a target sequence within said targetpolynucleotide, wherein said guide sequence is linked to a tracr matesequence which in turn hybridizes to a tracr sequence.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a CRISPR complex to bind to the polynucleotidesuch that said binding results in increased or decreased expression ofsaid polynucleotide; wherein the CRISPR complex comprises a CRISPRenzyme complexed with a guide sequence hybridized or hybridizable to atarget sequence within said polynucleotide, wherein said guide sequenceis linked to a tracr mate sequence which in turn hybridizes to a tracrsequence. Similar considerations and conditions apply as above formethods of modifying a target polynucleotide. In fact, these sampling,culturing and re-introduction options apply across the aspects of thepresent invention.

Indeed, in any aspect of the invention, the CRISPR complex may comprisea CRISPR enzyme complexed with a guide sequence hybridized orhybridizable to a target sequence, wherein said guide sequence may belinked to a tracr mate sequence which in turn may hybridize to a tracrsequence. Similar considerations and conditions apply as above formethods of modifying a target polynucleotide.

Kits

In one aspect, the invention provides kits containing any one or more ofthe elements disclosed in the above methods and compositions. Elementsmay be provided individually or in combinations, and may be provided inany suitable container, such as a vial, a bottle, or a tube. In someembodiments, the kit includes instructions in one or more languages, forexample in more than one language.

In some embodiments, a kit comprises one or more reagents for use in aprocess utilizing one or more of the elements described herein. Reagentsmay be provided in any suitable container. For example, a kit mayprovide one or more reaction or storage buffers. Reagents may beprovided in a form that is usable in a particular assay, or in a formthat requires addition of one or more other components before use (e.g.in concentrate or lyophilized form). A buffer can be any buffer,including but not limited to a sodium carbonate buffer, a sodiumbicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, aHEPES buffer, and combinations thereof. In some embodiments, the bufferis alkaline. In some embodiments, the buffer has a pH from about 7 toabout 10. In some embodiments, the kit comprises one or moreoligonucleotides corresponding to a guide sequence for insertion into avector so as to operably link the guide sequence and a regulatoryelement. In some embodiments, the kit comprises a homologousrecombination template polynucleotide. In some embodiments, the kitcomprises one or more of the vectors and/or one or more of thepolynucleotides described herein. The kit may advantageously allows toprovide all elements of the systems of the invention.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. The present examples, along with the methodsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses which are encompassed withinthe spirit of the invention as defined by the scope of the claims willoccur to those skilled in the art.

Example 1: Particle-Mediated Delivery of CRISPR-Cas9 Components intoHematopoietic Stem Cells (HSCs)

Applicants have demonstrated that Cas9 can be delivered to cells viaparticles. In this instance, in that many nucleic therapeutics mayrequire the delivery of both the one or more sgRNA and the Cas9 nucleaseconcurrently, we demonstrated the ability to deliver in this fashion.

The sgRNA was pre-complexed with the Cas9 protein, before formulatingthe entire complex in a particle. Twenty different particle formulationswere generated and tested for CRISPR-Cas9 delivery efficiency intocells. Each formulation was made with a different molar ratio of fourcomponents known to promote delivery of nucleic acids into cells:1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC), polyethyleneglycol (PEG), and cholesterol (e.g. DOTAP:DMPC:PEG:Cholesterol MolarRatios; Formulation number 1=DOTAP 100, DMPC 0, PEG 0, Cholesterol 0;Formulation number 2=DOTAP 90, DMPC 0, PEG 10, Cholesterol 0;Formulation number 3=DOTAP 90, DMPC 0, PEG 5, Cholesterol 5).

Particles were formed using an efficient, multistep process. First, Cas9protein and sgRNA targeting the gene EMX1 or the control gene LacZ weremixed together at a 1:1 molar ratio at room temperature for 30 minutesin sterile, nuclease free 1×PBS. Separately, DOTAP, DMPC, PEG, andcholesterol were dissolved in 100% ethanol. The two solutions were mixedtogether to form particles containing the Cas9-sgRNA complexes. Afterthe particles were formed, HSCs in 96 well plates were transfected with15 ug Cas9 protein per well. Three days after transfection, HSCs wereharvested, and the number of insertions and deletions (indels) at theEMX1 locus were quantified.

The invention is further described by the following numbered paragraphs:

1. A method of modifying an organism or a non-human organism bymanipulation of a target sequence in a hematopoetic stem cell (HSC),wherein the target sequence has an association with a mutation having anassociation with an aberrant protein expression or with a diseasecondition or state, wherein “normal” comprises wild type, and “aberrant”comprises expression that gives rise to a condition or disease state,said method comprising:

-   -   delivering to an HSC a non-naturally occurring or engineered        composition comprising:        -   I. a CRISPR-Cas9 system guide sequence RNA that hybridizes            to a target nucleotide sequence in the HSC, or a            polynucleotide encoding the guide sequence, operably for            transcription in the HSC, and        -   II. a Cas9 enzyme, optionally comprising at least one or            more nuclear localization sequences, or a polynucleotide            encoding the Cas9 enzyme, optionally comprising at least one            or more nuclear localization sequences, operably for            expression in the HSC,

to obtain an HSC population,

wherein the guide sequence directs sequence-specific binding of a CRISPRcomplex to the target nucleotide sequence in the HSC, and the CRISPRcomplex comprises the Cas9 enzyme complexed with the guide sequence;

optionally the method including also delivering a polynucleotidetemplate, wherein the template provides expression of a normal or lessaberrant form of a protein; and/or

optionally the method including, prior to the delivering, isolating orobtaining HSC from the organism or non-human organism, and/or

optionally the method including expanding the HSC population to obtainmodified HSCs, and/or

optionally the method including administering HSCs from the HSCpopulation or modified HSCs to the organism or non-human organism.

2. The method of numbered paragraph 1 wherein the target sequencecomprises a DNA sequence.

3. The method of numbered paragraphs 1, or 2 wherein the guide sequenceRNA comprises a chimeric RNA (chiRNA) polynucleotide including a tracrmate sequence and a tracr sequence, or the polynucleotide encoding theguide sequence include coding for including the tracr mate sequence andthe tracr sequence, operably for transcription in the HSC, and in theCRISPR complex the tracr mate sequence hybridizes to the tracr sequence.

4. The method of any one of numbered paragraphs 1, 2 or 3 wherein thedelivering comprises delivery of

-   -   I. the CRISPR-Cas9 system guide, or the polynucleotide encoding        the guide sequence, and at least one or more tracr mate        sequences or at least one or more polynucleotides encoding the        at least one or more tracr mate sequences operably for        transcription in the HSC, or the polynucleotide encoding the        guide sequence including at least one or more polynucleotides        encoding the at least one or more tracr mate sequences operably        for transcription in the HSC,    -   II. the Cas9 enzyme, optionally comprising at least one or more        nuclear localization sequences, or the polynucleotide encoding        the Cas9 enzyme, optionally comprising at least one or more        nuclear localization sequences, and    -   III. tracr sequence or a polynucleotide sequence encoding the        tracr sequence operably for transcription in the HSC, and        in the CRISPR complex the tracr mate sequence hybridizes to the        tracr sequence.

5. The method of any one of numbered paragraphs 1-4 wherein deliveringcomprises delivery of

-   -   I. the CRISPR-Cas system guide sequence RNA.

6. The method of any one of numbered paragraphs 1-5 wherein deliveringcomprises delivery of

-   -   II. the Cas9 enzyme, optionally comprising at least one or more        nuclear localization sequences.

7. The method of any one of numbered paragraphs 1-6 wherein deliveringcomprises delivery of

-   -   I. the polynucleotide encoding the guide sequence.

8. The method of any one of numbered paragraphs 1-7 wherein deliveringcomprises delivery of

-   -   II. the polynucleotide encoding the Cas9 enzyme, optionally        comprising at least one or more nuclear localization sequences.

9. The method of numbered paragraph 8 wherein delivering comprisesdelivery of the guide sequence RNA comprises the chimeric RNA (chiRNA)polynucleotide including the tracr mate sequence and the tracr sequence.

10. The method of numbered paragraph 9 wherein delivering comprisesdelivery of the polynucleotide encoding the guide sequence includecoding for including the tracr mate sequence and the tracr sequence,operably for transcription in the HSC, and in the CRISPR complex thetracr mate sequence hybridizes to the tracr sequence.

11. The method of any preceding numbered paragraph wherein deliveringcomprises delivery of

-   -   II. the Cas9 enzyme, optionally comprising at least one or more        nuclear localization sequences.

12. The method of any preceding numbered paragraph wherein deliveringcomprises delivery of

-   -   II. the polynucleotide encoding the Cas9 enzyme, optionally        comprising at least one or more nuclear localization sequences.

13. The method of any preceding numbered paragraph wherein thedelivering comprises delivery of

-   -   I. the CRISPR-Cas system guide sequence and at least one or more        tracr mate sequences or at least one or more polynucleotides        encoding the at least one or more tracr mate sequences.

14. The method of any preceding numbered paragraph wherein thedelivering comprises delivery of:

-   -   I. the CRISPR-Cas system guide sequence and at least one or more        tracr mate sequences.

15. The method of any preceding numbered paragraph wherein thedelivering comprises delivery of

-   -   I. the polynucleotide encoding the CRISPR-Cas system guide        sequence and at least one or more tracr mate sequences or at        least one or more polynucleotides encoding the at least one or        more tracr mate sequences.

16. The method of any preceding numbered paragraph wherein thedelivering comprises delivery of

-   -   III. the a polynucleotide sequence encoding the tracr sequence        operably for transcription in the HSC.

17. The method of any preceding numbered paragraph wherein the targetsequence is on a strand of a polynucleotide duplex, and method obtainscleavage of the polynucleotide duplex whereby on one strand there is a5′ overhang and on the other strand there is a 3′ overhang.

18. The method of any one of numbered paragraphs 1-16 wherein the targetsequence is a first target sequence on a first strand of thepolynucleotide duplex and there is a second target sequence on a secondstrand of the polynucleotide duplex, and the method comprises deliveryof a first CRISPR-Cas system guide sequence RNA that hybridizes to thefirst target nucleotide sequence in the HSC, or a polynucleotideencoding the first guide sequence, operably for transcription in theHSC, and delivery of a second CRISPR-Cas system guide sequence RNA thathybridizes to the second target nucleotide sequence in the HSC, or apolynucleotide encoding the second guide sequence, operably fortranscription in the HSC.

19. The method of numbered paragraph 18 wherein the delivery comprisesdelivering the first guide sequence.

20. The method of numbered paragraph 18 wherein the delivery comprisesdelivering the polynucleotide encoding the first guide sequence.

21. The method of any one of numbered paragraphs 18-20 wherein thedelivery comprises delivering the second guide sequence.

22. The method of any one of numbered paragraphs 18-20 wherein thedelivery comprises delivering the polynucleotide encoding the secondguide sequence.

23. The method of any one of numbered paragraphs 18-22 wherein either orboth of the first or second guide sequence comprises a tracr matesequence and a tracr sequence and the guide comprises a chimeric RNA(chiRNA) polynucleotide sequence.

24. The method of any one of numbered paragraphs 1-16 wherein the targetsequence is a first target sequence on a first strand of thepolynucleotide duplex and there is a second target sequence on a secondstrand of the polynucleotide duplex, and the method comprises deliveryof: a first CRISPR-Cas system guide sequence RNA that hybridizes to thefirst target nucleotide sequence in the HSC including at least one ormore tracr mate sequences, or a polynucleotide encoding the first guidesequence and at least one or more tracr mate sequences, operably fortranscription in the HSC; and a second CRISPR-Cas system guide sequenceRNA that hybridizes to the second target nucleotide sequence in the HSCincluding at least one or more tracr mate sequences, or a polynucleotideencoding the second guide sequence including at least one or more tracrmate sequences, operably for transcription in the HSC; and a tracrsequence or a polynucleotide encoding the tracr sequence, operably fortranscription in the HSC.

25. The method of numbered paragraph 24 wherein the delivery comprisesdelivering the first CRISPR-Cas system guide sequence RNA including atleast one or more tracr mate sequences.

26. The method of numbered paragraph 25 wherein the delivery comprisesdelivering the polynucleotide encoding the first guide sequence and atleast one or more tracr mate sequences, operably for transcription inthe HSC.

27. The method of any one of numbered paragraphs 24-26 wherein thedelivery comprises delivering the second CRISPR-Cas system guidesequence RNA including at least one or more tracr mate sequences.

28. The method of any one of numbered paragraphs 24-26 wherein thedelivery comprises delivering the polynucleotide encoding the secondguide sequence including at least one or more tracr mate sequences,operably for transcription in the HSC.

29. The method of any one of numbered paragraphs 24-26 wherein thedelivery comprises delivering the tracr sequence.

30. The method of any one of numbered paragraphs 24-26 wherein thedelivery comprises delivering the polynucleotide encoding the tracrsequence, operably for transcription in the HSC.

31. The method of any of the preceding numbered paragraphs wherein theCas9 is a nickase

32. The method of any of the preceding numbered paragraphs wherein theCRISPR-Cas system guide sequence RNA comprises an sgRNA.

33. The method of any of the preceding numbered paragraphs wherein thetarget sequence is associated with a genomic locus of interestassociated with the mutation.

34. The method of any of the preceding numbered paragraphs whereinoperably for transcription in the HSC includes the polynucleotidesequence operably linked to a regulatory element for expression ortranscription of the polynucleotide sequence.

35. The method of any of the preceding numbered paragraphs wherein whenthere is a polynucleotide(s) encoding (a) the guide sequence, (b) thetracr mate sequence and (c) the tracr sequence, (a), (b) and (c) arearranged in a 5′ to 3′ orientation.

36. The method of any preceding numbered paragraph wherein the organismor non-human organism is a mammal.

37. The method of numbered paragraph 36 wherein the mammal is a human.

38. The method of any preceding numbered paragraph wherein when theCRISPR complex arises from delivery of polynucleotide(s) for allcomponents of the CRISPR complex.

39. The method of numbered paragraph 38 wherein the polynucleotide(s)are delivered via a single vector or particle.

40. The method of numbered paragraph 38 wherein the polynucleotide(s)are delivered via more than one vector.

41. The method of numbered paragraph 38 wherein the polynucleotide(s)are delivered via a single vector.

42. The method of any preceding numbered paragraph wherein delivery isvia one or more particles contacting the HSC, wherein the one or moreparticles contain CRISPR complex component(s) and/or polynucleotide(s)therefor.

43. The method of numbered paragraph 42 wherein the method comprisescontacting the HSC with a particle containing the CRISPR complex.

44. The method of any one of numbered paragraphs 1-42 wherein deliveryis via one or more vectors contacting the HSC or one or more particlescontaining one or more vectors contacting the HSC, wherein the one ormore vectors contain the polynucleotide(s) encoding CRISPR complexcomponent(s).

45. The method of numbered paragraph 44 wherein the method comprisescontacting the HSC with a vector containing the polynucleotide(s)encoding all CRISPR complex components, or a particle containing such avector.

46. The method of any one of numbered paragraphs 44-45 wherein thevector comprises a viral, plasmid or nucleic acid molecule vector.

47. The method of any one of numbered paragraphs 42-46 whereinparticle(s) is/are formed by a method comprising or consistingessentially of or consisting of admixing (i) a mixture of the CRISPRcomplex components with (ii) a mixture comprising or consistingessentially of or consisting of surfactant, phospholipid, biodegradablepolymer, lipoprotein and alcohol, whereby particle(s) containing theCRISPR-complex are formed.

48. The method of numbered paragraph 47 wherein the mixture (i) includesthe polynucleotide template.

49. The method of any one of numbered paragraphs 1-47 including alsodelivering the polynucleotide template.

50. The method of numbered paragraph 48 or 49 wherein the delivery ofthe polynucleotide template is via the template being included in aparticle containing the CRISPR complex.

51. The method of numbered paragraph 50 wherein the delivery of thepolynucleotide template is via a particle.

52. The method of numbered paragraph 51 wherein the particle containingthe polynucleotide template is separate from particle(s) containingCRISPR complex component(s) and/or polynucleotide(s) coding therefor.

53. The method of numbered paragraph 49 wherein the delivery of thepolynucleotide template is via a vector.

54. The method of numbered paragraph 53 wherein the vector is separatefrom vector(s) containing polynucleotide(s) coding for CRISPR complexcomponent(s).

55. The method of numbered paragraph 53 wherein the vector is the sameas the vector containing polynucleotide(s) coding for CRISPR complexcomponents.

56. The method of any one of the preceding numbered paragraphs whereinthe polynucleotide encoding the CRISPR protein is codon optimized forexpression in the HSC.

57. The method of any one of the preceding numbered paragraphs whereinthe Cas9 comprises one or more mutations in a catalytic domain.

58. The method of numbered paragraph 57, wherein the one or moremutations comprise, with reference to SpCas9, D10A, E762A, H840A, N854A,N863A or D986A, or an analogous mutation in a CRISPR protein other thanSpCas9.

59. The method of numbered paragraph 58 wherein the mutation comprises,with reference to SpCas9, a D10A mutation, or an analogous mutation in aCas9 other than SpCas9.

60. The method of any one of numbered paragraphs 1-58 wherein the Cas9comprises an SaCas9.

61. The method of any preceding numbered paragraph including, prior tothe delivering, isolating or obtaining HSC from the organism ornon-human organism.

62. The method of any preceding numbered paragraph including expandingthe HSC population to obtain modified HSCs.

63. The method of numbered paragraph 62 including administering HSCsfrom the HSC population or modified HSCs to the organism or non-humanorganism.

64. The method of any one of the preceding numbered paragraphs whereinthe target or the genomic locus of interest is associated withHemophilia B, sickle cell anemia, SCID, SCID-X1, ADA-SCID, Hereditarytyrosinemia, β-thalassemia, X-linked CGD, Wiskott-Aldrich syndrome,Fanconi anemia, adrenoleukodystrophy (ALD), metachromatic leukodystrophy(MLD), HIV/AIDS, Krabbe Disease, Polycythemia vera (PCV),myeloproliferative neoplasm, Familial essential thrombocythaemia (ET) orAlpha-mannosidosis.

65. The method of any one of the preceding numbered paragraphs whereinthe target or the genomic locus of interest is associated with anImmunodeficiency disorder, Hematologic condition, a Leukodystrophy orgenetic lysosomal storage disease.

66. Use of a CRISPR-Cas complex as defined in any of the foregoingnumbered paragraphs in the preparation of a medicament for a patient inneed of treatment for a disease condition or state, comprisingperforming a method as in any of the foregoing numbered paragraphs.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention.

1. A method of modifying an organism or a non-human organism bymanipulation of a target sequence in a hematopoetic stem cell (HSC),wherein the target sequence has an association with a mutation having anassociation with an aberrant protein expression or with a diseasecondition or state, wherein “normal” comprises wild type, and “aberrant”comprises expression that gives rise to a condition or disease state,said method comprising: delivering to an HSC a non-naturally occurringor engineered composition comprising: I. a CRISPR-Cas9 system guide thathybridizes to a target nucleotide sequence in the HSC, or apolynucleotide encoding the guide sequence, operably for transcriptionin the HSC, and II. a Cas9 enzyme, comprising at least one or morenuclear localization sequences, or a polynucleotide encoding the Cas9enzyme; comprising at least one or more nuclear localization sequences,operably for expression in the HSC, to obtain an HSC population, whereinthe guide directs sequence-specific binding of a CRISPR complex to thetarget nucleotide sequence in the HSC, and the CRISPR complex comprisesthe Cas9 enzyme complexed with the guide; optionally the methodincluding also delivering a polynucleotide template, wherein thetemplate provides expression of a normal or less aberrant form of aprotein; and/or optionally the method including, prior to thedelivering, isolating or obtaining HSC from the organism or non-humanorganism, and/or optionally the method including expanding the HSCpopulation to obtain modified HSCs, and/or optionally the methodincluding administering HSCs from the HSC population or modified HSCs tothe organism or non-human organism.
 2. The method of claim 1, whereinthe target sequence is on a strand of a polynucleotide duplex, and themethod obtains cleavage of the polynucleotide duplex whereby on onestrand there is a 5′ overhang and on the other strand there is a 3′overhang.
 3. The method of claim 1, wherein the target sequence is afirst target sequence on a first strand of the polynucleotide duplex andthere is a second target sequence on a second strand of thepolynucleotide duplex, and the method comprises delivery of a firstCRISPR-Cas system guide that hybridizes to the first target nucleotidesequence in the HSC, or a polynucleotide encoding the first systemguide, operably for transcription in the HSC, and delivery of a secondCRISPR-Cas system guide that hybridizes to the second target nucleotidesequence in the HSC, or a polynucleotide encoding the second systemguide, operably for transcription in the HSC.
 4. The method of claim 3,wherein either or both of the first or second guide comprises a tracrmate sequence and a tracr sequence and the guide comprises a chimericRNA (chiRNA) polynucleotide sequence.
 5. The method of claim 1, whereinthe target sequence is a first target sequence on a first strand of thepolynucleotide duplex and there is a second target sequence on a secondstrand of the polynucleotide duplex, and the method comprises deliveryof: a first CRISPR-Cas system guide that hybridizes to the first targetnucleotide sequence in the HSC including at least one or more tracr matesequences, or a polynucleotide encoding the first system guide and atleast one or more tracr mate sequences, operably for transcription inthe HSC; and a second CRISPR-Cas system guide that hybridizes to thesecond target nucleotide sequence in the HSC including at least one ormore tracr mate sequences, or a polynucleotide encoding the secondsystem guide including at least one or more tracr mate sequences,operably for transcription in the HSC; and a tracr sequence or apolynucleotide encoding the tracr sequence, operably for transcriptionin the HSC.
 6. The method of claim 1, wherein the Cas9 is a nickase. 7.The method of claim 1, wherein the CRISPR-Cas system guide comprises ansgRNA.
 8. The method of claim 1, wherein the target sequence isassociated with a genomic locus of interest associated with themutation.
 9. The method of claim 1, wherein operably for transcriptionin the HSC includes the polynucleotide sequence operably linked to aregulatory element for expression or transcription of the polynucleotidesequence.
 10. The method of claim 1 wherein the organism or non-humanorganism is a mammal.
 11. The method of claim 10, wherein the mammal isa human.
 12. The method of claim 1, wherein the non-naturally occurringor engineered composition is delivered via a single vector or particle.13. The method of claim 1, wherein the composition is delivered via morethan one vector.
 14. The method of claim 1, wherein delivery is via oneor more particles contacting the HSC, wherein the one or more particlescontain CRISPR complex component(s) and/or polynucleotide(s) therefor.15. The method of claim 1, wherein delivery is via one or more vectorscontacting the HSC or one or more particles containing one or morevectors contacting the HSC, wherein the one or more vectors contain thepolynucleotide(s) encoding CRISPR complex component(s).
 16. The methodof claim 15 wherein the vector comprises a viral, plasmid or nucleicacid molecule vector.
 17. The method of claim 15, wherein particle(s)is/are formed by a method comprising or consisting essentially of orconsisting of admixing (i) a mixture of the CRISPR complex componentswith (ii) a mixture comprising or consisting essentially of orconsisting of surfactant, phospholipid, biodegradable polymer,lipoprotein and alcohol, whereby particle(s) containing theCRISPR-complex are formed, and wherein the mixture (i) optionallyincludes a polynucleotide template.
 18. The method of claim 1, whereinthe Cas9 comprises one or more mutations in a catalytic domain.
 19. Themethod of claim 18, wherein the one or more mutations comprise, withreference to SpCas9, D10A, E762A, H840A, N854A, N863A or D986A, or ananalogous mutation in a CRISPR protein other than SpCas9.
 20. The methodof claim 1, wherein the target sequence is associated with Hemophilia B,sickle cell anemia, SCID, SCID-X1, ADA-SCID, Hereditary tyrosinemia,β-thalassemia, X-linked CGD, Wiskott-Aldrich syndrome, Fanconi anemia,adrenoleukodystrophy (ALD), metachromatic leukodystrophy (MLD),HIV/AIDS, Krabbe Disease, Polycythemia vera (PCV), myeloproliferativeneoplasm, Familial essential thrombocythaemia (ET) orAlpha-mannosidosis, or wherein the target sequence is associated with anImmunodeficiency disorder, Hematologic condition, a Leukodystrophy orgenetic lysosomal storage disease.